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Leukemia Leukemia Document Transcript

  • Molecular Biology Methods Flowcytometric and FISH Markers inChildhood Acute Lymphoblastic Leukemia Dr Gihan EL Hussieny Gawish, MSc, PhD.
  • 2 Acknowledgements First of all, I cannot give a word to fulfill my deeps love and thanksto (Allah) for lighting me the way not only throughout this piece of work butalso throughout my whole life.This work is dedicated to:My husband; Dr Hussein Al Omer and my FamilyI am indebted to King Saud University for support and encouragement tofinish this work.Also, I wish to express my deep thanks to:  Prof. Dr./ Abdelfattah M. Attalah, Professor of Genetics & Immunology, George Washington University, USA (Former). Director of Biotechnology Research Center, New Damietta, Egypt, for continuous advice  Prof. Dr./ Ahmed Abd Al Salam Settin, Professor of Pediatrics & Genetics, Faculty of Medicine, Mansoura University for his continuous help Finally, I am indebted to all the team of the honorable Genetics Unit, Mansoura University Children Hospital, for their continuous support and encouragement Gihan El Hussieny Gawish January 2009
  • 3 Contents Title PageIntroduction 1Review of Literature 4 I- Acute lymphoblastic leukemia 4 1-Definition 4 2-Incidence of Leukemia 5 3-Types of leukemia 6 4-Biological Classification of ALL 6 5-Causing of Leukemia 7 6-The Signs of Leukemia 9 7-Stages of Childhood ALL 11 8-Treatment of Childhood ALL 11 9-Four Phases of Treatment 14 II- Cell cycle and apoptosis 15 1-Cell cycle 15 1-1-Cell cycle and cancer 19 2-Apoptosis and its markers 20 2-1-The mechanism of apoptosis 21 2-2-Apoptosis-targeted therapies for hematologica malignancies 25 2-3-The apoptosis promoter (p53) 30 2-4-The inhibitor of apoptosis 35 2-4-1-Bcl2 proteins 35 2-4-2-C-myc oncogene 38 III-Flow cytometry 42 1-Introduction 42 2-Principles of flow cytometric instrumentation 44 2-1-Fluidic system 46 2-2-Illumination system 49 2-3-Optical and electronics system 53 2-4-Data storage and computer control system 54 3-Data analysis 59 IV- Applications of flow cytometry 63 1-Cell cycle analysis 65 1-1-Staining procedure 66
  • 4 Page 1-2-Evaluation of DNA histogram 67 2-Immunophenotyping Applications 71 2-1-Erythrocyte analysis 72 2-2-HIV monitoring 73 2-3-Immunophenotyping of leukemias 73 2-4Quantification of stem cells 75 2-5-Platelet analysis 75 2-6-Testing for HLA-B27 76 3-Major applications of apoptosis analysis 76 3-1- Apoptosis light scatter 77 3-2-Apoptosis DNA analysis 78 3-3-Apoptosis cell membrane analysis 80 3-4-Apoptosis enzyme analysis 82 3-5-Apoptosis organelle analysis 83 4- Detection of apoptotic markers 83V- Flourescence in situ hybridization 86 1-Introduction 86 2-Three different types of FISH probes 91 2-1-Locus specific probes 91 2-2-Alphoid or centromeric repeat probes 91 2-3-Whole chromosome probes 92 3-Applications of FISH 92 3-1-ALL investigation by FISH 96 3-1-1-Philadelphia 97VI- References 106VII- Life Flowcytometric Figures 144VIII-Life FISH Pictures 147
  • 5 List of Figure Review of Literature PageFigure (2-1) A schematic representation of the mammalian cell cycle 18Figure (2-2) The intrinsic or mitochondrial pathway 22Figure (2-3) The mechanism of apoptosis (Apoptosis triggered by 24 external signals: the extrinsic or death receptor path way)Figure (2-4) Diagram of the mitochondrial and death receptor pathways 26 of cell deathFigure (3-1) Facscaliblur flow cytometry instrument 47Figure (3-2) Flow cytometer system (Facscalibur) 48Figure (3-3) Flow cytometers use the principle of hydrodynamics 50 focusing for presenting cells to a laserFigure (3-4) A simplified illustration of Flow Cytometry 52Figure (3-5) Two parameter histogram and dot plot displaying FL1-FITC 57 on the x axis and FL2-PE on the y axisFigure (3-6) FlowJo program 58Figure (3-7) Analysis pulse width versus pulse height or area we can 60 eliminate the majority of G0 doublets that appear as G2Figure (3-8) DNA histogram 61Figure (3-9) DNA histogram (aneuphliod population) 62Figure (4-1) Coeffecient of Variation (C.V.) 69Figure (4-2) Propidium iodide and TO-PRO-3 79Figure (4-3) Sub G1 peak by propidium iodide staining 81Figure (5-1) Fluoresence in situ hypridization 88
  • 6 List of AbbreviationsAL Acute leukemiaALL Acut lymphoblastic leukemiaALT Alanine amino transaminaseAO Acridine orangeAST Aspartate amino transaminaseBM Bone marrowCBC Complete blood pictureCD Cluster of differentiationCML Chronic myeloid leukemiaCV Coefficient of variationDAPI 4-6 diamino -2-2phenylindoleDI DNA indexDMSO DimethylsulfoxideDNA Deoxyribonucleic acidEB Ethedium bromideEDTA Ethyline diamine tetraacetic acidFAB French American BritishFACS Flow activated cell sorterFISH Fluorescence in situ hybridizationFITC Flourecien isothiocyanateG0/G1 Phase represents the gap in of DNA replication time between mitosis and the startG2/M Phase represents the gap between the end of DNA replication onest of mitosisHIV Human immunodeficiency virusHLA Human leukocyte antigenHPV Human papilloma virusLC Liver cirrhosisMMC MithramycinMRD Minimal residual diseasePBS Phosphate buffer salinePI Propidium iodidePS PhosphatidylserineS phase DNA syntheisWBC White blood cells
  • 7 Introduction Childhood acute lymphoblastic leukemia (ALL) is a disease in whichtoo many underdeveloped lymphocytes are found in a childs blood and bonemarrow. Lymphocytes are infection-fighting white blood cells. ALL is themost common form of leukemia in children, and the most common kind ofchildhood cancer (Moorman et al., 2006). Acute lymphoblastic leukemia (ALL) represents nearly one third ofall pediatric cancers. Annual incidence of ALL is about 30 cases per millionpopulations, with a peak incidence in patients aged 2-5 years. Although a smallpercentage of cases are associated with inherited genetic syndromes, the causeof ALL remains largely unknown (Jeffrey, 2005). Flow cytometry can be applied in basic research and in the clinic toidentify and measure apoptotic cells. The choice of a particular flow methoddepends on several variables (cell system, type of flow cytometer, type ofapoptosis inducer, type of information required) (Bogh and Duling, 2005). The cell cycle was subdivided into four consecutive phases; G1 orpre-synthetic phase, S, G2 or post-synthetic phase, and M phase during whichmitotic division into two daughter cells takes place. The G2 phase representsthe gap in time between the end of DNA replication and onset of mitosis. It ispossible to discrimination between G1 vs, S vs, G2 or M cells because of thedifference in their DNA content (Rabinovitch, 1993). The DNA content of the cell can provide a great deal of informationabout the cell cycle. The measurement of the DNA content of cells was oneof the first major applications of flow cytometry (Albro et al., 1993).
  • 8 Apoptosis (programmed cell death) is a physiologicphenomenon where in the dying cell plays an active part in its own destruction(Schuler et al., 1994). Apoptosis plays a role in many diseases. There is agreat potential for treatment of these diseases in developing agents that canalter the apoptotic process and change the natural disease progression.Molecules whose roles in apoptosis have been investigated include Bcl-2 andc-myc proteins, the p53 tumor suppressor gene and various tumor suppressorgene products (Menendez et al., 2004). P53 is a pro-apoptotic genes present in all cells, but has specialsignificance to cancer cells. It is a tumor repressor gene, meaning that itspresence reduces the occurrence of cancer tumors by promoting apoptosis incancer cells (Polyak et al., 1997). BCL2 is an important regulator of apoptosis,The oncogenic activity of the Bcl2 gene is carried out via suppression oflymphocytic apoptosis or programmed cell death (Cory & Adams, 2002 andRoumier et al., 2002). C-Myc is widely known as a crucial regulator of cellproliferation in normal and neoplastic cells (Wechsler et al., 1997& Facchiniand Penn, 1998). The technology of flow cytometry and the discovery of a method toproduce monoclonal antibodies have made possible the clinical use of flowcytometry for the identification of cell populations. Monoclonal antibodies(tagged) with the fluorescent dye are commonly used for the identification ofcell surface antigens and fluorescent dyes that directly and specifically bind tocertain components of the cell (i.e. DNA) are used for cell cycle analysis(Zhang et al., 2005). Fluorescence in situ hybridization (FISH) allows identification ofspecific sequences in a structurally preserved cell, in metaphase or interphase
  • 9 (Chatzimeletiou et al., 2005). FISH is increasingly used for theidentification of ALL. FISH plays an important role in detecting chromosomechanges (Primo et al., 2003). Almost all the chromosome abnormalities in ALL are translocations.The most common one is Philadelphia chromosome. It is the main product ofthe t(9;22) translocation. This translocation causes a rearrangement betweenthe proto-oncogene c-ABL and a gene called the breakpoint cluster region(BCR). The BCR/ABL fusion gene resulting from t(9;22) translocation. FISHis increasingly used for the identification of BCR/ABL gene rearrangements(Rudolph et al., 2005). І- Acute Lymphoblastic Leukemia
  • 01 Acute lymphoblastic leukemia (ALL) is the most common formof childhood cancer. It is a type of cancer that starts from white blood cells inthe bone marrow called lymphocytes. In most cases it quickly moves into theblood. It can then spread to other parts of the body including the lymph nodes,liver, spleen and central nervous system (Moorman et al., 2006). Leukemia is a cancer of the blood cells. There are several types ofleukemia and these are classified by how quickly they progress and what cellthey affect. Acute leukemia is fast-growing and can overrun the body within afew weeks or months. By contrast, chronic leukemia is slow-growing andprogressively worsens over years (Carolyn et al., 2002). Normal blood cells contain white blood cells, red blood cells, plateletsand fluid called plasma. All of these products are formed in the bone marrow,a spongy area located in the center of bones. It contains a small percentage ofcells that are in development and are not yet mature. These cells are calledblasts. Once the cell has matured, it moves out of the bone marrow and intothe circulating blood. The body has mechanisms to know when more cells areneeded and has the ability to produce them in an orderly fashion (Carroll etal., 2003).
  • 00 1-Incidence of Leukemia: Acute lymphoblastic leukemia is the most common form of childhoodleukemia where it accounts for about 75% of childhood leukemia and 25% ofall pediatric cancer (Lanzkowsky, 2000). National Cancer Institue, CairoUniversity, ALL represents 23.3% of all pediatric malignancies and 75% ofall pediatric leukemias. In a more recent research in the PediatricHeamatology/Oncology Unit, Ain Shams University Hospital, ALLconstitutes 82% of all leukemic cases (Khalifa et al., 1999). The global incidence of leukemias is about 8 to 9 per 100,000 peopleeach year. Approximately 250,000 new cases occur annually worldwide.Leukemia accounts for 2.5% of overall cancer incidence. However, itsincidence among children demonstrates its significance. Although childhoodcases (through 14 years of age) account for about 12% of all leukemias,childhood cancer is the second biggest killer of children (after accidents) andleukemia is the most common form of childhood cancer. The incidence ofchildhood ALL in the United States has increased approximately 20% over thepast two decades, mostly in the 0- to 4-year-old age group. Over the course ofthis century, leukemia rates have also generally increased (Sandler and Ross,1997). Acute lymphoblastic leukemia affects slightly more boys than girls.It occurs predominantly in children, peaking at four years of age. It is seenmore frequently in industrialized nations, and it is slightly more commonamong white children and boys. Studies have suggested that patients who areyounger than thirty five years of age far better than older patients (Jeffrey,2005).
  • 02 2-Types of leukemia: By considering whether leukemias are acute or chronic, and whetherthey are myelogenous or lymphocytic, they can be divided into four maintypes. The first one is an acute myeloid leukemia which occurs in both childrenand adults. The second one is an acute lymphocytic leukemia which is themost common type seen in children, but also seen in adults over65.The thirdone is a chronic myelogenous leukemia which occurs mostly in adults. Chroniclymphocytic is the fourth type which is the most often seen in people overage55, can affect younger adults, but almost never seen in children (Pui,1995). In acute leukemia, the bone marrow cells are unable to properlymature. Immature leukemia cells, which are often called blasts, continue toreproduce and accumulate. In chronic leukemia, the cells can mature but notcompletely. They are not really normal. They generally do not fight infectionas well as do normal white blood cells. Of course, they live longer, build up,and crowd out normal cells. The types of leukemia are also grouped by thetype of white blood cell that is affected, leukemia that affects lymphoid cellsis called lymphocytic leukemia, and leukemia that affects myeloid cells iscalled myeloid leukemia or myelogenous leukemia (Lichtman et al., 1995).3-Biological Classification of ALL: Acute lymphoblastic leukemia blasts are derived from either B-cell orT-cell lineages, as determined by cell surface and other markers. A smallpercentage of the cells are either so primitive that they do not express enoughmarkers to identify (Ross et al., 2003 and Pullen et al., 1999).
  • 03 Acute lymphoblastic leukemia is categorized according to asystem know as the French-American-British (FAB) MorphologicalClassification Scheme for ALL. ALL1 is mature-appearing lymphoblasts (T-cells or pre-B-cells); these cells are small with uniform genetic material,regular nuclear shape, nonvisible nucleoli, and little cytoplasm. ALL2 isimmature and pleomorphic lymphoblasts (T-cells or pre-B-cells), these cellsare large, variable in size, varaiable genetic material, irregular nuclear shape,one or more large nucleoli and variable cytoplasm. ALL3 is lymphoblast(B-cells),these are large, genetic material is finely stripped and uniform, nuclearshape is regular, there are one or more prominent nucleoli, and cytoplasm ismoderately abundant (Schrappe et al., 2000).4-Causing of Leukemia: The causes of the disease are not known, but experts believe that ALLdevelops from a combination of genetic and environmental factors. A numberof genetic mutations associated with ALL have been identified. Missing ordefective genes that suppress tumors are responsible for cases of ALL (Guo etal., 2005). Several things have been identified as risk factors-that is, exposure tothem puts a person at a higher risk of developing leukemia, but it is not acertainly that this exposure will lead to leukemia. These factors includeexposure to high-energy radiation, like that released from a nuclear accidentor bomb. Some genetic syndrome put a person at higher risk. People who workwith the chemical benzene over a long period of time also have a greaterchance of getting leukemia. Some scientist feel that exposure toelectromagnetic fields, like those that come from power lines, may put aperson to higher risk, but this has not been proven (Pui et al., 2001).
  • 04 Heredity, radiation, chemical exposures, and treatment withchemotherapeutic agents have been implicated in the development ofleukemia. Viral infection by at least one known virus, human T-cellleukemia/lymphotropic virus type I (HTLV-1), is a well-understood cause ofadult T-cell leukemia (Franchini, 1995 and Greaves, 1997). Another group of risk factors includes occupational andenvironmental exposure to radiation or chemicals. The best established causeof leukemia among children is in utero exposure to diagnostic X-rays.Leukemia in adults is strongly associated with occupational exposure toionizing radiation. There is little evidence, however, that nonionizing radiationsuch as electromagnetic fields (EMF) induces leukemia. Indeed, two recentstudies have shown that EMF exposure is not a major risk factor for leukemiain children or in adults. Some studies have reported an association betweencancer and high levels of electromagnetic radiation (EMR). Whether lowerlevels of radiation (eg, living near power lines, video screen emissions, smallappliances, cell phones) play any major role is uncertain but probably unlikely(Linet et al., 1997 and Verkasalo, 1996). Because most people in the general population are not exposed tochemotherapeutic drugs or occupationally exposed to radiation or chemicalsolvents, exposure to these agents cannot explain the causes of the majority ofleukemia cases diagnosed each year. We conservatively estimate that thecauses of at least 20,000 (approximately 70%) of the 28,000 new leukemiacases that develop annually in the United States are unexplained. Thus, thecauses of leukemia remain largely unknown. Although some success has beenachieved in treating leukemias, especially in children, mortality rates haveremained relatively high (approximately 75% in the United States) (Kazak etal., 1997).
  • 05 Genetic predisposition may play a major role in both adult andchildhood leukemia. Although the Leukemia Society of America emphasizesthe fact that anyone may develop the disease, an increased risk exists amongEastern European Jews, and a decreased risk exists among Asians (differencesin diet and lifestyle may play a role, however). Individuals with a familyhistory of leukemia or lymphoma have a 5.6-fold increased risk for AML.Parents of children with Down syndrome also have an increased risk ofleukemia (Greaves, 1997 and Shannon et al., 1992). Up to 65% of leukemias contain genetic rearrangements, calledtranslocations, in which some of the genetic material (genes) on achromosome may be altered, or shuffled, between a pair of chromosomes. Forexample the most common genetic injury in ALL is t(12;21), which means atranslocation with a genetic shift between chromosome 12 and 21. It is alsoreferred to as TEL-AML1 fusion and occurs in approximately 20% of ALLpatients. Researchers believe that this translocation may occur during fetaldevelopment in some patients. About 20% of adults and about 5% of childrenwith ALL have a genetic abnormality called the Philadelphia (Ph)chromosome t(9;22). Another important chromosome translocation is t(4;11)involving the MLL gene on chromosome II. Often occurring in children underone year old (Khandakar et al., 2005).5-The Signs and diagnosis of Leukemia: The blast cells are unable to perform their normal function of fightinginfection, so patients may develop fevers or infections that wont go away. Asthe number of immature cells (blasts) increases, the normal cells are crowdedout. This leads to low red blood cell counts and platelets (Smith et al., 1996).
  • 06 Acute lymphoblastic leukemia tends to cause symptoms morerapidly than chronic leukemia. Some common symptoms include fever, chills,weakness and fatigue, swollen or tender lymph nodes, liver or spleen, easybleeding or bruising, swollen or bleeding gums, night sweats, and bone pain.The abnormal cells can accumulate in the brain or spinal cord, causingheadaches, vomiting, confusion, or seizures (Adachi et al., 2005). In acute lymphoblastic leukemia, the doctor asks about medicalhistory and conducts a physical exam. During the exam, abnormalities such asenlarged spleen, liver or lymph nodes may be detected, prompting furtherinvestigation. Complete blood count would find blast cells present in theblood, thus suggestion a diagnosis of leukemia. This test can reveal that thepatient has leukemia. A sample of bone marrow is determined the type ofleukemia (Champlin et al. 1989 and Burger et al., 2003). A complete blood cell count is the first step in diagnosing ALL. Thistest will often show various findings, including the following: The presence ofcirculatory leukemic blast cells, the presence and severity of anemia and thecount of a variety of blood cell types. (A high white blood cell count indicatesa more severe disease.) These tests will not always show the presence ofleukemic cells. Blood tests do not always detect leukemia, and about 10% ofpatients with ALL have a normal blood cell count (Adachi et al., 2005). If the results of the blood tests are abnormal or the physician suspectsleukemia despite normal cell counts, a bone marrow aspiration and biopsy arethe next steps (Rezaei et al., 2003). If bone marrow examination confirms ALL, a spinal tap may beperformed, which uses a needle inserted into the spinal canal. A sample of
  • 07 cerebrospinal fluid with leukemia cells is a sign that the disease hasspread to the central nervous system. In most cases of childhood ALL,leukemic cells are not found in the cerebrospinal fluid (Vieira et al., 2005).6-Treatment of Childhood ALL: The treatment depends on age, the results of laboratory tests, andwhether or not the child has been previously treated for leukemia. UntreatedALL means that no treatment has been given except to reduce symptoms.There are too many white blood cells in the blood and bone marrow, and theremay be other signs and symptoms of leukemia. Remission means thattreatment has been given and the number of white blood cells and other bloodcells in the blood and bone marrow is normal that there no signs or symptomsof leukemia. Recurrent disease means that the leukemia has come back aftergoing into remission. Refractory disease means that the leukemia failed to gointo remission following treatment (Bassan et al., 1997). There are treatments for all patients with childhood acutelymphoblastic leukemia. The primary treatment for ALL is chemotherapy.Radiaion therapy may be used in certain cases. Bone marrow transplantationis being studied in clinical trials (Uckun et al., 1997). Acute lymphoblastic leukemia patients should receive chemotherapydrugs as soon as possible after diagnosis. Chemotherapy uses strong drugs tokill leukemia cells. The goal of chemotherapy is to achieve remission (nosymptoms of ALL) and to restore normal blood cell production. Commonchemotherapy drugs include doxorubicin, fludarabine and cyclophosphamide.The drugs used depend on factors such as the patients age and the number andtype of leukemia cells in the blood. Unfortunately, chemotherapy also kills
  • 08 normal cells, so ALL patients receiving chemotherapy may have sideeffects, including nausea, tiredness and a higher risk of infections (Balduzzi etal., 2005). For most patients, chemotherapy restores normal blood cellproduction within a few weeks, and microscopic examinations of their bloodand marrow samples will show no signs of leukemia cells. When this happens,the disease is in remission. Although chemotherapy often brings long-lastingremissions in children, in adults, ALL frequently returns. If the ALL returns,patients and their doctors can consider more chemotherapy or a marrow orblood cell transplant. Chemotherapeutic agents kill cancer cells by activatingapoptosis, or programmed cell death. Major apoptotic pathways and thespecific role of key proteins in this response is described. The expression levelof some of these proteins, such as Bcl2, BAX, and caspase 3, has been shownto be predictive of ultimate outcome in hematopoietic tumors. New therapeuticapproaches that modulate the apoptotic pathway are now available and may beapplicable to the treatment of childhood ALL (Donadieu & Hill, 2001 andNakase et al., 2005). Radiation therapy uses X-rays or other high-energy rays to kill cancercells and shrink tumors. Radiation for ALL usually comes from a machineoutside the body (external beam radiation therapy) (Durrant et al., 1997). Bone marrow transplantation is a newer type of treatment. First, highdoses of chemotherapy with or without radiation therapy are given to destroyall of the bone marrow in the body. A bone marrow transplant using marrowfrom a relative or person not related to the patient is called an allogeneic bonemarrow transplant (Ulrich et al., 2001).
  • 09 An even newer type of bone marrow transplant, called autologousbone marrow transplant, is being studied in clinical trials. During thisprocedure, bone marrow is taken from the patient and may be treated withdrugs to kill any cancer cells. The marrow is frozen to save it. The patient isthen given high-dose chemotherapy with or without radiation therapy todestroy all of the remaining marrow. The frozen marrow that was saved isthawed and given through a needle in a vein to replace the marrow that wasdestroyed (Sebban et el., 1994). Treatment outcome is dependent not only on the therapy applied, butimportantly, also on the underlying biology of the tumor and the host. Each ofthese variables must be factored into initial treatment decisions, as well as laterrefinements based on initial response, and several biological features. It isrecognized that with improvements in therapy, certain variables might losetheir prognostic value; therefore, risk assignment plans should be routinelyreassessed. Finally an optimal system should allow for comparison of theoutcomes of similar or identical patients, treated on different protocols (Choiet al., 2005). There are generally four phases of treatment for ALL. The first phase,remission induction therapy, uses chemotherapy to kill as many of theleukemia cells as possible to cause the cancer to go into remission. The secondphase, called central nervous system (CNS) prophylaxis, is preventive therapy,it involves using intrathecal and/or high-dose systemic chemotherapy to theCNS to kill any leukemia cells present there. It is also used to prevent thespread of cancer cells to the brain and spinal cord even if no cancer has beendetected there. Radiation therapy to the brain may also be given, in addition tochemotherapy, for this purpose. CNS prophylaxis is often given in conjunction
  • 21 with consolidation therapy. Once a child goes into remission and there nosigns of leukemia, a third phase of treatment called consolidation orintensification therapy, is given. Consolidation therapy uses high-dosechemotherapy to attempt to kill any remaining leukemia cells. The fourthphase of treatment, called maintenance therapy, uses chemotherapy for severalyears to maintain the remission (Attal et al., 1995).
  • 20 II- Cell cycle and Apoptosis1-Cell cycle: The concept of the cycle in its current form is introduced by Howardand Plec, (1953). They observed that DNA synthesis (S- phase) in individualcells was discontinuous and occupied a discrete portion of the cell life and wasconstant in duration. Mitotic division (M-phase) was seen to occur after certainperiod of time following DNA replication. A distinct phase between DNAreplication and mitosis was also apparent (Look et al., 1996). Cell cycle phase of G1 was historically considered to be a time cellshad little observable activity. Since this time precedes DNA synthesis, the termGap 1 (G1) was coined. They have diploid chromosome (2C=46chromosome). At a certain point in the cells life, the DNA syntheticmachinery turns on. This phase of the cells life is labeled "S" for synthesis.As the cell proceeds through this phase, its DNA content increases from 2C to4C. At the end of S, the cell has duplicated its genome and it is in the tetraploidstate. After the S phase, the cell again enters a phase that was historicallythought to be quiescent. Since this phase is the second gap region, it is referredto as G2. In the G2 phase, the cell is producing the necessary proteins that willplay a major role in cytokinase. After a highly variable amount of time, thecell enters mitosis (M). DNA content remains constant at 4C until the cellactually divides at the end telophase (Liblit, 1993). The process of replicating DNA and dividing a cell can be describedas a series of coordinated events that compose a "cell division cycle,"illustrated for mammalian cells in Fig (2-1). In each cell division cycle,chromosomes are replicated once (DNA synthesis or S-phase) and segregated
  • 22 to create two genetically identical daughter cells (mitosis or M-phase).These events are spaced by intervals of growth and reorganization (gap phasesG1 and G2). Cells can stop cycling after division, entering a state of quiescence(G0). Commitment to traverse an entire cycle is made in late G1. At least twotypes of cell cycle control mechanisms are recognized: a cascade of proteinphosphorylations that relay a cell from one stage to the next and a set ofcheckpoints that monitor completion of critical events and delay progressionto the next stage if necessary (Nasmyth, 1996). The first type of control involves a highly regulated kinase family.Kinase activation generally requires association with a second subunit that istransiently expressed at the appropriate period of the cell cycle; the periodic"cyclin" subunit associates with its partner "cyclin-dependent kinase" (CDK)to create an active complex with unique substrate specificity. Regulatoryphosphorylation and dephosphorylation fine-tune the activity of CDK-cyclincomplexes, ensuring well-delineated transitions between cell cycle stages(Elledge, 1996). A second type of cell cycle regulation, checkpoint control, is moresupervisory. It is not an essential part of the cycle progression machinery. Cellcycle checkpoints sense flaws in critical events such as DNA replication andchromosome segregation. When checkpoints are activated, for example byunderreplicated or damaged DNA, signals are relayed to the cell cycle-progression machinery. These signals cause a delay in cycle progression, untilthe danger of mutation has been averted. Because checkpoint function is notrequired in every cell cycle, the extent of checkpoint function is not as obviousas that of components integral to the process, such as CDKs (Sherr, 1996).
  • 23Figure (2-1): A schematic representation of the mammalian cell cycle(Nasmyth, 1996).
  • 24 1-1-Cell cycle and cancer: Superficially, the connection between the cell cycle and cancer isobvious: cell cycle machinery controls cell proliferation, and cancer is adisease of inappropriate cell proliferation. Fundamentally, all cancers permitthe existence of too many cells. However, this cell number excess is linked ina vicious cycle with a reduction in sensitivity to signals that normally tell a cellto adhere, differentiate, or die. This combination of altered properties increasesthe difficulty of deciphering which changes are primarily responsible forcausing cancer (Jacks and Weinberg, 1996). The first genetic alterations shown to contribute to cancerdevelopment were gain-of-function mutations. These mutations define a set of"oncogenes" that are mutant versions of normal cellular "protooncogenes."The products of protooncogenes function in signal transduction pathways thatpromote cell proliferation. However, transformation by individual oncogenescan be redundant (mutation of one of several genes will lead to transformation)or can be cell type-specific (mutations will transform some cells but have noeffect on others). This suggests that multiple, distinct pathways of geneticalteration lead to cancer, but that not all pathways have the same role in eachcell type (White, 1996). More recently, the significance of loss-of-function mutations incarcinogenesis has become increasingly apparent. Mutations in these so-called"tumor suppressor" genes were initially recognized to have a major role ininherited cancer susceptibility. Because inactivation of both copies of a tumorsuppressor gene is required for loss of function, individuals heterozygous formutations at the locus are phenotypically normal. Thus, unlike gain-of-function mutations, loss-of-function tumor suppressor mutations can be carried
  • 25 in the gene pool with no direct deleterious consequence. However,individuals heterozygous for tumor suppressor mutations are more likely todevelop cancer, because only one mutational event is required to preventsynthesis of any functional gene product (Morgenbesser et al., 1994). It now appears that tumor suppressor gene mutations are highly likelyto promote, and may even be required for, a large number of spontaneous aswell as hereditary forms of cancer. Loss of function of the tumor suppressorgene product pRb, for example, would be predicted to liberate E2Ftranscriptional activators without requiring phosphorylation and thus bypass anormal negative regulation controlling entry into the cycle. Loss of the tumorsuppressor gene product p16 would have a similar consequence, liberatingE2Fs by increasing pRb phosphorylation . In addition, cell cycle progressioncan be halted at several points by the tumor suppressor gene product p53,activated in response to checkpoints sensing DNA and possibly alsochromosome damage; loss of p53 would remove this brake to cycling(Symonds et al., 1994).2-Apoptosis and its markers: Apoptosis and necrosis are too distinct, mutually exclusive, modes ofcell death. Apoptosis, frequently referred to as programmed cell death is anactive and physiological mode of cell death, in which the cell itself designsand executes the program of its own demise and subsequent body disposal.Different patterns of apoptosis (early and delayed apoptosis) many cell types,cells of hematopoietic origin in particular, undergo apoptosis rapidly, to withinfew hours following exposure to relatively high concentration of cytotoxicagents (Majino and Joris, 1995).
  • 26 Apoptosis can be defined as gene-directed cellular self-destruction although this is really a phenomenon where cells are programmedto die at a particular point, e.g. during embryonic development, and even herecells may go through an apoptotic pathway. However, apoptosis is certainly adistinct process from other forms of oncosis leading to necrosis (Gerbaulet etal., 2005 and Wallach et al., 1999). Apoptosis affects individual cells, physiological induction e.g. lack ofsignals, phagocytosis by macrophages or other cells and there is noinflammatory response. Necrosis affects group of cells, non physiologicalinduction e.g. virus and poison, phagocytosis of macrophages and there isinflammatory response (Wirth et al., 2005). There are three different mechanisms by which a cell commits suicideby apoptosis. In the intrinsic or mitochondarial pathway, the outer membranesof mitochondria in a healthy cell express the protein; Bcl2 on their surface.Bcl2 is bound to a molecule of the protein Apaf-1. Internal damage to the cell(e.g., from reactive oxygen species) causes Bcl2 to release Apaf-1; a relatedprotein, Bax, to penetrate mitochondrial membranes causing cytochrome c toleak out. The released cytochrome c and Apaf-1 bind to molecules of caspase9 Fig. (2-2). The resulting complex of cytochrome c, Apaf-1, caspase 9 andATP is called the apoptosome. The apoptosome aggregate in the cytosol (Niuet al., 2005 and Lam et al., 2005 and Kroemer& Reed 2000).
  • 27Figure (2-2): The intrinsic or mitochondrial pathway (Lam et al., 2005).
  • 28 Caspase 9 is one of a family of over a dozen caspases. They areall proteases. They get their name because they cleave proteins-mostly eachother at aspiratic acid residues. Caspase 9 cleaves and, in so doing, activatesother caspases. The sequential activation of one caspase by another creates anexpanding cascade of proteolytic activity (rather like that in blood clotting andcomplement activation) which leads to digestion of structural proteins in thecytoplasm, degradation of chromosomal DNA and phagocytosis of the cell(Wada et al., 2005). In the extrinsic or death receptor pathway, Fas and the TNF receptorare integral membrane proteins with their receptor domains exposed at thesurface of the cell. Binding of the complementary death activator (FasL andTNF respectively) transmits a signal to the cytoplasm that leads to activationof caspase 8. Caspase 8 (like caspase 9) initiates a cascade of caspaseactivation leading to phagocytosis of the cell Fig. (2-3). For example,cytotoxic T cells recognize (bind to) their target, they produce more FasL attheir surface, this binds with the Fas on the surface of the target cell leading toits death by apoptosis. In some cases, final destruction of the cell is guarantedonly withits engulfment by a phagocyte (Bijangi et al., 2005 and Vega et al.,2005). In the third way, neurons, and perhaps other cells, have another wayto self-destruct that unlike the two paths described above, doesnt use caspase.Apoptosis- inducing factor (AIF) is a protein that is normally located in theinter membrane space of mitochondaria. When the cell receives a signal tellingit that it is time to die, AIF is released from the mitochondrial, it is migratesinto the nucleus and binds to DNA, Which triggers the destruction of the DNAand cell death (Urbano et al., 2005).
  • 29Figure (2-3): The mechanism of apoptosis (Apoptosis triggered byexternal signals: the extrinsic or death receptor path way) (Bijangi et al.,2005).
  • 31 Defects in programmed cell death (apoptosis) mechanisms playimportant roles in the pathogenesis and progression of hematologicalmalignancies, allowing neoplastic cells to survive beyond their normallyintended life-spans and subverting the need for exogenous survival factors.Apoptosis defects also serve as an important complement to proto-oncogeneactivation, as many deregulated oncoproteins that drive cell division alsotrigger apoptosis (Evan and Littlewood, 1998). Similarly, errors in DNA repair and chromosome segregationnormally trigger cell suicide as a defense mechanism for eradicatinggenetically unstable cells, and thus apoptosis defects permit survival of thegenetically unstable cells, providing opportunities for selection ofprogressively aggressive clones (Ionov et al., 2000). Chemotherapy and irradiation trigger apoptosis in tumor cells and anunderstanding of the biochemical pathways involved in apoptosis provides anopportunity to classify tumors based on their response to common inductionregimens. Multiple distinct signaling pathways regulate apoptosis, but twomajor cell death pathways have been implicated in hematologicalmalignancies: the mitochondrial pathway and the death receptor pathway Fig.(2-4) (Evans et al., 2002).
  • 30Figure (2-4): Diagram of the mitochondrial and death receptor pathwaysof cell death (Evans et al., 2002).
  • 32 Both of these pathways ultimately activate members of thecaspase family of proteins that are responsible for executing the terminalphases of apoptosis. p53 protein levels rise in response to various cellularstresses including chemotherapy. p53 induces the loss of mitochondrialmembrane potential with subsequent release of cytochrome c, which forms acomplex, the "apoptosome," with the adapter molecule Apaf-1, ATP, andcaspase-9. This complex, in turn, activates caspase-3 (Evans et al., 2002). Another proximal pathway of cell death involves death receptorsignaling at the cell surface. Binding of CD95-L and other tumor necrosisfactor (TNF) family ligands to their death inducing receptors, CD95/APO-1/FAS or TNF- and TRAIL respectively, leads to receptor trimerization andthe recruitment of adapter molecules. These molecules include FADD/MORT-1 that in turn lead to recruitment and activation of caspase-8. This initiatorcaspase also cleaves and activates downstream caspases, including caspase-3.Although generally described as being distinct, these two proximal pathwaysare interconnected. For example, caspase-8 cleaves the pro-apoptotic proteinBID, which results in translocation to the mitochondria and release ofcytochrome c (Kishi et al., 2003, Blom, 2000, de Franchis et al., 2000 andGoto et al., 2001). Several studies have examined the prognostic significance ofapoptotic protein expression in leukemia. Defects in the p53 pathway aredistinctly rare in childhood malignancies including ALL, where mutations aredetected in < 5% of cases at the time of initial diagnosis. However, relapsedblasts may harbor mutations of p53 gene much more commonly. Further, ALLblasts at relapse have been noted to express high levels of the Mdm-2 protein,which abrogates p53 signaling (Dirven et al., 1995 and Pemble et al., 1994).
  • 33 Cancer-associated defects in apoptosis play a role inchemoresistance and radioresistance, increasing the threshold for cell death,and thereby requiring higher doses for tumor killing (Tschopp et al., 1999 andMakin et al., 2000). Melanoma (skin cancer) cells avoid apoptosis by inhibiting theexpression of the gene encoding Apaf-1. Some cancer cells, especially lungand colon cancer cells, secrete elevated levels of a soluble (decoy) moleculethat binds to FasL, plugging it up so it cannot bind Fas. Thus cytotoxic T cells(CTL) cannot kill the cancer cells by the mechanism of death receptorpathway. Other cancer cells express high levels of FasL, and can kill anycytotoxic T cells (CTL) that try to kill them because CTL also express Fas (butare protected from their own FasL) (Meijer et al., 2005). Apoptosis plays a role in many diseases, such as cancer, viralinfections, and autoimmune and neurodegenerative disorders. There is a greatpotential for treatment of these diseases in developing agents that can alter theapoptotic process and change the natural disease progression. Moleculeswhose roles in apoptosis have been investigated include Bcl-2 and c-mycproteins, the p53 tumor suppressor gene and various tumor suppressor geneproducts, MAP kinases, and proteases (Menendez et al., 2004).2-1-The apoptosis promoter (p53): p53 stimulates a wide network of signals that act through two majorapoptotic pathways. The extrinsic, death receptor pathway triggers theactivation of a caspase cascade, and the intrinsic, mitochondrial pathway shiftsthe balance in the Bcl-2 family towards the pro-apoptotic members, promotingthe formation of the apoptosome, and consequently caspase-mediated
  • 34 apoptosis. The impact of these two apoptotic pathways may be enhancedwhen they converge through Bid, which is a p53 target. The majority of theseapoptotic effects are mediated through the induction of specific apoptotictarget genes. However, p53 can also promote apoptosis by a transcription-independent mechanism under certain conditions. Thus, a multitude ofmechanisms are employed by p53 to ensure efficient induction of apoptosis ina stage-, tissue- and stress-signal-specific manner (Linda & Carol, 1996 andSusan et al., 2003). Some cancer causing viruses use tricks to prevent apoptosis of thecells they have transformed. Several human papilloma viruses (HPV) havebeen implicated in causing cervical cancer. One of them produces a protein(E6) that binds and inactivates the apoptosis promoter p53. Binding of Fasligand or agonistic anti-Fas antibody to the death receptor Fas can activate acaspase-cascade resulting in apoptosis. Fas cell surface expression wasdetermined by flow cytometry (Hougardy et al., 2005). Genes involved in apoptosis are either pro-apoptotic (promoteapoptosis) or anti-apoptotic (inhibit apoptosis). P53 is a pro-apoptotic genespresent in all cells, but has special significance to cancer cells. It is a tumorrepressor gene, meaning that its presence reduces the occurrence of cancertumors by promoting apoptosis in cancer cells. Normally it induces apoptosisby activating caspases 9, 8, 7, and 3. The loss of p53 decreases caspaseactivation and therefore the cell will not undergo apoptosis. Mutation in thep53 gene is the most common mutation in cancer; it is present in about half ofall cancer tumors, 80% in all colon cancer tumors, 50% of lung cancer tumors,and 40% of breast cancer tumors (Polyak et al., 1997).
  • 35 Under normal conditions p53 is a short-lived protein. The p53inhibitor Mdm2 (Hdm2 in humans) is largely responsible for keeping p53 inthis state. Mdm2 inhibits the transcriptional activity of p53 and, moreimportantly, promotes its degradation by the proteasome (Levine, 1997). p53 mutants in tumours have a reduced affinity for DNA and areduced ability to induce apoptosis. We describe a mutant with the oppositephenotype, an increased affinity for some p53-binding sites and an increasedability to induce apoptosis. The apoptotic function requires transcriptionactivation by p53 (Elisabeth et al., 1999). Early observations suggested that p53 may function as an oncogene,because overexpression of p53 appeared to cause oncogenic transformation ofcells. In the late 1980s, however, several critical discoveries defined thenormal function of p53 to be anti-oncogenic. Wild-type p53 genes, whenintroduced into cells, were found to be growth suppressive (Isabela et al.,2000). p53 plays multiple roles in cells. Expression of high levels of wild-type (but not mutant) p53 has two outcomes: cell cycle arrest or apoptosis. Inresponse to genotoxic stress, p53 acts as an "emergency brake" inducing eitherarrest or apoptosis, protecting the genome from accumulating excessmutations. Consistent with this notion, cells lacking p53 were shown to begenetically unstable and thus more prone to tumors (Isabela et al., 2000). p53 promotes cytochrome c release through the induction of targetgenes encoding BH3-only proteins. Importantly, p53 also induces APAF-1expression through a response element within the APAF-1 promoter (Kannanet al., 2001)
  • 36 The tumor suppressor gene product p53 is clearly a central playerin many biochemical pathways that are pivotal to human carcinogenesis. Thesequence-specific DNA binding properties of this nuclear phosphoproteinregulate the transcription of a continually expanding number of genes, theprotein products of which regulate cell cycle progression and apoptosis(Isabela et al., 2000). Loss of p53 function by mutation is common in cancer. However,most natural p53 mutations occur at a late stage in tumor development, andmany clinically detectable cancers have reduced p53 expression but no p53mutations. It remains to be fully determined what mechanisms disable p53during malignant initiation and in cancers without mutations that directlyaffect p53. p53 mutants in tumours have a reduced affinity for DNA and areduced ability to induce apoptosis (Niu et al., 2005). p53 expression has important clinical implications as an indicator ofprognosis and response to chemotherapy or radiotherapy in different humantumor types. The common effect of p53 mutations found in tumours is toinactivate p53 as a transcription factor. Consequently, a great deal of effort hasbeen expended in trying to identify transcriptional targets of p53. Particularattention has been paid to target genes which may mediate cell-cycle arrestand apoptosis (Ko and Prives, 1996 ). p53 dependent G1 and G2 arrest requires induction of the p21 cyclin-dependent kinase inhibitor. In contrast, no single gene can explain p53-induced apoptosis. Many p53 target genes have been identified which functionin known apoptotic pathways, regulate survival factor signalling, induceapoptosis when over expressed or are involved in biochemical events linkedto apoptosis (Buckbinder et al., 1997 , Miyashita and Reed, 1995 , Owen-
  • 37 Schaub et al., 1995, Polyak et al., 1997, Varmeh-Ziaie et al., 1997,McCurrach et al., 1997 and Rampino et al., 1997). p53 can activate target genes through a non-canonical sequence. Thefirst such example is in the p53-induced gene 3 (PIG3), which has beenimplicated in the accumulation of reactive oxygen species and apoptosisinduction (Polyak et al., 1997). Another recently described example is the geneencoding the pro-apoptotic phosphatase PAC1, which is induced throughbinding of p53 to a novel palindromic binding site (Yin et al., 2003). A novel insight into the interplay between p53 and its familymembers, p63 and p73, in the induction of apoptosis has been recently revealed(Flores et al., 2002). The effect of p63 and p73 on p53 transcriptional activity,using a selection of knockout mouse embryo fibroblasts (MEFs), defined twodistinct classes of target gene. Whereas p53 alone is sufficient for the inductionof p21 and Mdm2, the induction of the apoptotic genes PERP, Bax and Noxarequires p53 together with p63 and p73. This finding demonstrates an essentialrole for both p63 and p73 in the efficient induction of apoptotic target genes byp53. The mechanism of this cooperation is currently unknown, but it mayinvolve an enhanced binding to and/or stabilization of the transcriptioncomplex on the promoters of p53 apoptotic target genes by the cooperativeaction of all three members (Urist and Prives, 2002). In addition to the contribution of p63 and p73 to the apoptotic functionof p53, they play an important role in the precise control of cell death duringnormal mouse development. p73 also plays a role in the induction of cell deathin response to DNA damage, a process involving cooperation between the Abltyrosine kinase and p73 (Shaul, 2000).
  • 38 Immunohistochemical (IHC) detection of p53 expression hasbeen established as a relatively easy and straightforward method for fresh andarchival tissues. Available monoclonal antibodies recognize both wild-typeand mutant forms, but there may be a selective detection of the latter owing tothe very short half-life of the former (Porter et al., 1992 and Soussi et al.,1994). p53 is a tumor suppressor that is rarely mutated in ALL patients butwhose function is frequently altered by mutations to genes that code forproteins that regulate p53 function. Activation of p53 occurs in response tocells that have acquired DNA damage that may be engaged in aberrant cellproliferation. Mutations to proteins that regulate p53 function, like HDM2,p14, and p21, are frequent findings in ALL (Roman et al., 2002). Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblastcell lines were analyzed via flow cytometry (FCM) for expression of p53protein along with their DNA content. Significantly elevated levels of the p53protein was present in some but not all of the transformed cell lines.Quantitation of p53 protein in cell lines containing BPV-1 DNA revealed thatthe tumorigenic cell lines expressed higher levels of the p53 protein (Agrawalet al., 1994). The correlation between p53 abnormalities and DNA aneuploidy andthat analysis of p53 protein is useful for prediction of clinical course inesophageal squamous cell carcinoma (Goukon et al., 1994). Liu et al., (2004) evaluated changes in apoptotic proteins expressionthat occur in response to chemotherapy in pediatric cases with acute leukemiajust prior to and 1, 6 and 24 hours following the administration of multiagent
  • 39 chemotherapy. They found great heterogeneity in the patterns of apoptoticprotein expression in the initial response to chemotherapy among individualpatient samples. Importantly, no increases in p53, p21 or Mdm-2 proteinexpression were seen in leukemic blasts from the standard risk patients whoseinitial treatment consisted of the non-p53-dependent drugs, vincristine andprednisone. In the subgroup of children who received at least one p53 dependentdrug, patients could be segregated into two groups, one group that showed up-regulation of p53 protein and its target p21, and another group that showed noincrease following therapy, thus identifying at least two distinct pathwaysleading to apoptosis (Chen et al., 1996).2-2-Bcl2 proteins Members of the Bcl-2 protein family play pivotal roles in thedecision and execution phases of apoptosis in the mitochondrial pathway. Todate, 24 Bcl-2 family members have been identified as either pro- (e.g., Bax,Bak, Bcl-XS, Bid, Bad, and Noxa) or anti- (e.g., Bcl-2 and Bcl-XL) apoptoticproteins. Bcl-2 proteins form homo- and heterodimeric complexes to regulatemitochondrial channel formation and subsequent release of cytochrome c fromthe mitochondria (Kishi et al., 2003, Blom, 2000, de Franchis et al., 2000,Goto et al., 2001 and Cryns et al., 1999). The Bcl2 family proteins are the central regulators of the mitochondrialpathway. Bcl2 is an inhibitor of apoptosis. Bcl2 and its human homologintroduce a new category of oncogenes that act by decreasing cell death. Overexpression of Bcl2 promotes oncogensis by repressing cell death and
  • 41 extending cell life. However, overexpression can also lead to retardationof cell cycling via prolongation of the G1 phase of the cycle (Webb et al., 2005and Green & Reed, 1998). The Bcl2 family of intracellular proteins is the central regulator ofcaspase activation, and its opposing factions of anti- and pro-apoptoticmembers arbitrate the life-or-death decision. The oncogenic activity of theBcl2 gene is carried out via suppression of lymphocytic apoptosis orprogrammed cell death. (Cory & Adams, 2002 and Roumier et al., 2002). BCL2 is an important regulator of apoptosis, first identified from itsinvolvement in follicular B cell lymphoma, where the common t(8:14)translocation causes the activation of the BCL2 oncogene. BCL2 is nowrecognised as a survival factor for many types of cell, notably neurons.Expression of BCL2 is widespread during embryogenesis but is restricted tolong-lived cells in the adult. A critical mediator of BCL2 apoptosis isinterleukin-1 beta-converting enzyme (ICE) a cysteine protease that processesIL-1 beta during the inflammatory response (Roumier et al., 2002). BCL2 is a member of a multigene family (highly conservedevolutionarily with viral homologues). Other proteins in the family (BCLX,BAD, BAX, BAD etc) antagonise inhibition of apoptosis by binding to BCL2.Hence the balance of various members of the BCL family determines theextent to which cell death is promoted or prevented. This model is consistentwith the findings of high levels of BCL2 in a variety of solid tumours (Jiangand Milner, 2003). Apoptosis can also be induced by a variety of cytokines e.g. TGF betafamily, which inhibit the proliferation of a wide variety of cell types that may
  • 40 undergo concomitant cell death. TGF beta induced apoptosis is blockedin myeloblastic leukaemia cells by BCL2 expressed at a level that does notblock but merely delays p53-induced apoptosis. This may reflect the fact thatboth TGF beta and p53 suppress BCL2 but only p53 has the ability to activateBAX, thus deflecting the expression pattern towards apoptosis (Seckin et al.,2002). Active cell suicide (apoptosis) is induced by events such as growthfactor withdrawal and toxins. It is controlled by regulators, which have eitheran inhibitory effect on programmed cell death (anti-apoptotic) or block theprotective effect of inhibitors (pro-apoptotic). Many viruses have found a wayof countering defensive apoptosis by encoding their own anti-apoptosis genespreventing their target-cells from dying too soon. All proteins belonging to theBcl-2 family contain either a BH1, BH2, BH3, or BH4 domain. All anti-apoptotic proteins contain BH1 and BH2 domains, some of them contain anadditional N-terminal BH4 domain (Bcl-2, Bcl-x (L), Bcl-w), which is neverseen in pro-apoptotic proteins, except for Bcl-x(S). On the other hand, all pro-apoptotic proteins contain a BH3 domain (except for Bad) necessary fordimerization with other proteins of Bcl-2 family and crucial for their killingactivity, some of them also contain BH1 and BH2 domains (Bax, Bak). TheBH3 domain is also present in some anti-apoptotic protein, such as Bcl-2 orBcl-x (L). Proteins that are known to contain these domains include vertebrateBcl-2 (alpha and beta isoforms) and Bcl-x (isoforms (Bcl-x(L) and Bcl-x(S))(Poliseno et al., 2002). Antiapoptotic B cell leukemia/lymphoma (BCL2) family proteinsare expressed in many cancers, but the circumstances under which theseproteins are necessary for tumor maintenance are poorly understood. A novelfunctional assay that uses Bcl2 homology domain (BH3) peptides to predict
  • 42 dependence on antiapoptotic proteins was exploiteded , a strategy, BH3profiling. BH3 profiling accurately predicts sensitivity to Bcl2 antagonistABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3profiling also accurately distinguishes myeloid cell leukemia sequence 1(MCL1) from Bcl2 dependence in myeloma cell lines. It was shown that thespecial sensitivity of CLL cells to Bcl2 antagonism arises from therequirement that Bcl2 tonically sequester proapoptotic BIM in CLL. ABT-737displaced BIM from Bcl2s BH3-binding pocket, allowing BIM to activateBAX, induce mitochondrial permeabilization, and rapidly commit the CLLcell to death. It was demonstrated that Bcl2 expression alone does not dictatesensitivity to ABT-737. Instead, Bcl2 complexed to BIM is the critical targetfor ABT-737 in CLL. An important implication is that in cancer, Bcl2 may noteffectively buffer chemotherapy death signals if it is already sequesteringproapoptotic BH3-only proteins. Indeed, activator BH3-only occupation ofBcl2 may prime cancer cells for death, offering a potential explanation for themarked chemosensitivity of certain cancers that express abundant Bcl2, suchas CLL and follicular lymphoma (Del Gazio et al., 2007). The relationship between gene expression of Bcl 2 and Bax and thetherapeutic effect in oral cancer patients had investigated. A significantcorrelation between Bcl-2/Bax gene expression ratio in the peripheral bloodmononuclear cells (PBMCs) from the patients, and the therapeutic effect ofradiation therapy These findings suggested that Bcl-2 and Bax gene expressionin PBMCs may be useful as a prognostic factor in oral cancer patients(Oshikawa et al., 2006). Epstein-Barr virus (EBV), the cause of mononucleosis and cause ofBurkitts lymphoma produces a protein similar to Bcl2 and produces anotherprotein that causes the cell to increase its own production of Bcl2. Both these
  • 43 actions make the cell more resistant to apoptosis (thus enabling the cancercell to continue to proliferate). Even cancer cells produced without theparticipation of viruses may have tricks to avoid apoptosis (Lu et al., 2005). Some B-cell leukemias and lymphomas express high levels of Bcl2,thus blocking apoptotic signals they may receive. The high levels result froma translocation of the Bcl2 gene into an inhancer region for antibodyproduction (Menendez et al., 2004). Bcl2-L12 contributes to the classical tumor biological features ofGlioblastoma (GBM) such as intense apoptosis resistance and florid necrosis,and may provide a target for enhanced therapeutic responsiveness of this lethalcancer (Stegh et al., 2007).2-3C-myc oncogene: The c-myc gene was discovered as the cellular homolog of the retroviral v-myc oncogene 20 years ago. The c-myc proto-oncogene wassubsequently found to be activated in various animal and human tumors. Itbelongs to the family of myc genes that includes B-myc, L-myc, N-myc, and s-myc; however, only c-myc, L-myc, and N-myc have neoplastic potential(Wechsler et al., 1997 and Facchini & Penn, 1998). Targeted homozygousdeletion of the murine c-myc gene results in embryonic lethality, suggestingthat it is critical for development. Homozygous inactivation of c-myc in ratfibroblasts caused a marked prolongation of cell doubling time, furthersuggesting a central role for c-myc in regulating cell proliferation (Mateyak etal., 1997). Bovine papillomavirus type 1 (BPV-1)-transformed mousefibroblast cell lines were analyzed via flow cytometry (FCM) for expression
  • 44 of c-myc protein along with their DNA content. Significantly elevatedlevels of the c-myc protein was present in some but not all of the transformedcell lines. Quantitation of c-myc protein in cell lines containing BPV-1 DNArevealed that the tumorigenic cell lines expressed higher levels of the c-mycprotein (Agrawal et al., 1994). The role of c-Myc in the cell cycle has been a confusing area due tothe collection of data from different experimental models, although it is wellestablished that c-myc is an early serum response gene. It should be noted thatmodels of serum or growth factor stimulation of starved cells primarily addressthe G0/G1 and G1/S transitions. Therefore, early studies implicated c-Myc inthe G0/G1 transition. In cycling cells, however, the participation of c-Myc inthe cell cycle may be different. Furthermore, in anchorage-dependent cellgrowth, c-Myc may affect other components of the cell cycle (Amati et al.,1998). It is proposed that c-Myc induces apoptosis through separate deathpriming and death triggering mechanisms in which death priming andmitogenic signals are coordinated. Investigation of the mechanisms thatunderlie the triggering steps may offer new therapeutic opportunities(Prendergast, 1999). The antiapoptotic effect of Epstein-Barr virus (EBV) was associatedwith a higher level of Bcl-2 expression and an EBV-dependent decrease insteady-state levels of c-MYC protein. Although the EBV EBNA-1 protein isexpressed in all EBV-associated tumors and is reported to have oncogenicpotential, enforced expression of EBNA-1 alone in EBV-negative Akata cellsfailed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the
  • 45 tumorigenic potential of Burkitt lymphoma and suggest a novel modelwhereby a restricted latency program of EBV promotes B-cell survival, andthus virus persistence within an immune host, by selectively targeting theexpression of c-MYC (Ingrid et al., 1999). Much recent research on c-Myc has focused on how it drivesapoptosis. c-Myc is widely known as a crucial regulator of cell proliferation innormal and neoplastic cells, but until relatively recently its apoptoticproperties, which appear to be intrinsic, were not fully appreciated. Its death-dealing aspects have gained wide attention in part because of their potentialtherapeutic utility in advanced malignancy, where c-Myc is frequentlyderegulated and where novel modalities are badly needed. Although its exactfunction remains obscure, c-Myc is a transcription factor and advances havebeen made in characterizing target genes which may mediate its apoptoticproperties (Hermeking, 2003). Ectopic expression of c-Myc (Myc) in most primary cell types resultsin programmed cell death, and malignant transformation cannot occur withoutadditional mutations that block apoptosis. The development of Myc-inducedlymphoid tumors was studied. Myc can be upregulated in acute myeloidleukemia (AML), but its exact role in myeloid leukemogenesis is unclear. Tostudy its role in AML, a murine stem cell virus (MSCV) retroviral genetransfer/transplantation system was used to broadly express Myc in the bonemarrow of mice either alone or in combination with antiapoptotic mutations.Myc expression in the context either of Arf/Ink4a loss or Bcl-2 coexpressioninduced a mixture of acute myeloid and acute lymphoid leukemias(AML+ALL). In the absence of antiapoptotic mutations however, all micetransplanted with MSCV-Myc developed AML exclusively. MSCV-Myc-induced AML was polyclonal, readily transplantable, possessed an intact Arf-
  • 46 p53 pathway, and did not display cytogenetic abnormalities by spectralkaryotyping analysis. Lastly, it was found that Myc preferentially stimulatedthe growth of myeloid progenitor cells in methylcellulose. These data providedthe first direct evidence that Myc is a critical downstream effector of myeloidleukemogenesis and suggested that myeloid progenitors are intrinsicallyresistant to Myc-induced (Hui et al., 2005). III-Flow cytometry
  • 47 Flow cytometry is a laser-based technology that is used to measurecharacteristics of biological particles. This technology is used to performmeasurements on whole cells as well as prepared cellular constituents such asnuclei and organelles (Melamed et al., 1990, Tileney et al., 1996 and McCoy,2002). The flow cytometer is an instrument for measuring scattered andfluorescent light from single particles. The physics of the interaction of lightwith single particles provides the scientific foundation for the design andoperation of the flow cytometer and for the critical evaluation of flowcytometric data (Scornik et al., 1994). Flow cytometry uses the principles of light scattering, light excitation,and emission of fluorochrome molecules to generate specific multi-parameterdata from particles and cells in the size range of 0.5um to 40um diameter. Cellsare hydro-dynamically focused in a sheath of phosphate buffer saline (PBS)before intercepting an optimally focused light source. Lasers are most oftenused as a light source in flow cytometry (Talbot, 1993). The technology of flow cytometry and the discovery of a method toproduce monoclonal antibodies have made possible the clinical use of flowcytometry for the identification of cell populations. Light scatter is utilized toidentify the cell populations of interest, while the measurement of fluorescenceintensity provides specific information about individual cells. Monoclonalantibodies (tagged) with the fluorescent dye are commonly used for theidentification of cell surface antigens and fluorescent dyes that directly andspecifically bind to certain components of the cell (i.e. DNA) are used for cellcycle analysis (Reckenwald, 1993 and Shapiro, 1995).
  • 48 Cells or particles are prepared as single-cell suspension for flowcytometric analysis. This allows them to flow single file in a liquid stream pasta laser beam. As the laser strikes the individual cells. First light scatteringoccurs that is directly related to structural and morphological cell features.Second, fluorescence occurs if the cells are attached to a fluorescent probe.Fluoresent probes are typically monoclonal antibodies that have beenconjucated to fluorochromes; they can also be fluorescent stains reagents thatare not conjugated to antibodies (Parks and Herzenberg, 1989 andRechenwald et al, 1993). Fluorescent probes are reacted with the cells or particles of interestbefore analysis; therefore, the amount of fluorescence emitted as a particlepasses the light source is proportional to the amount of fluorescent probebound to the cell or cellular constituent (Radcliff and Jaroszeski, 1998). After acquisition of light scattering and fluorescence data for eachparticle, the resulting information can be analyzed utilizing a computer andspecific software that are associated with the cytometer (Rose et al,. 1992 andLongobardi-Given, 1992). There are two distinct types of flow cytometers that can be used toacquire data from particles. One type can perform acquisition of lightscattering and fluorescence only. The other type is capable of acquiringscattering and fluorescence data but also has the powerful ability to sortparticles. Both types function in a similar manner during acquisition, forexample FACScan (Becton Dickinson), this equipped with an air –cooled 15mw argon ion laser emitting at 488 nm. Three fluorescence channels can bemeasured as well as two light scatter parameters. The FACScan is alsoequipped with a doublet discrimination module allowing the analysis of the
  • 49 cell cycle. The FACScan is user-operated (after instruction) and isavailable for use 24 hours per day (Kandathil et al., 2005). However, sorting instruments have the powerful ability to physicallyseparate particles based on light scattering and/or fluorescent emissioncharacteristics. Cytometers were originally designed to sort, for exampleFACS caliber 1, 2 (Becton Dickison), this used for analysis only. Unlike theFACScan which is a dual laser system. The primary laser is an air-cooled 15mw argon ion laser emitting at 488 nm thus allowing two light scatterparameters and three fluorescence channels to be measured. The second laseris ared diode laser emitting at 635nm. Thus allowing for the excitation of otherdyes such as allophycocyanin or to-pro-3 with power Macintosh G4 runningsystem 9.0 and cell Quest v 3.3. Thus, cytometers that perform acquisitionwithout sorting are the most common of the two types (Rose et al., 1992).1-Principles of flow cytometric instrumentation: Flow cytometers are very complex instruments that are composed offour closely related systems. The fluidic system transports particles from asuspension through the cytometer for interrogation by an illumination system.The resulting light scattering and fluorescence is collected, filtered, andconverted into electrical signals by the optical and electronics system. Thedata storage and computer control system saves acquired data and is also theuser interface for controlling most instrument functions. These four systemsprovide a very unique and powerful analytical tool for researchers andclinicians. This is because they analyze the properties of individual particles,and thousands of particles can be analyzed in a matter of seconds. Thus, datafor a flow cytometric sample are a collection of many measurements insteadof a single bulk measurement (Radcliff and Jarosezeski, 1998)..
  • 51 Histograms are the simplest modes of data representation.Histograms allow visualization of a single acquired parameter. Meanfluorescence and distributional statistics can be obtained based on markers thatthe user can graphically set on the plot. Multiple histograms can be overlaidon one another to depict qualitative and quantitative differences in two or moresamples. Two-parameter data plots are somewhat more complicated thanhistograms; however, they can yield more information. Two-parameter dotplots of FSC vs SSC allow visualization of both light-scattering parametersthat are important for identifying populations of interest. Bivariate fluorescentplots allow discrimination of dual-labeled populations that might remainhidden if histograms were used to display fluorescent data. Two-parameterplots that combine one light-scattering parameter and a fluorescent parameterare useful for analyzing control samples to elucidate the origin of nonspecificbinding. Data analysis is very graphically oriented. Experience and patternrecognition become important when using two-parameter data plots forqualitative as well as quantitative analysis. The technique of gating or drawingregions on dual parameter light-scatter plots allows one to exclude informationand examine the population of interest by disallowing particles that mightconfound or interfere with analysis. This is one of the fundamental uses forgating (Radcliff and Jarosezeski, 1998). Flow cytometers can be described as four interrelated systems whichare shown in Fig. (3-1). these four basic systems are common to all cytometersregardless of the instrument manufacturer and whether or not the cytometer isdesigned for analysis or sorting (Melamed et al., 1990 and Longobardi-Given,1992 Owens & Loken, 1995).1-1-Fluidic system:
  • 50 The fluidic system is the heart of a flow cytometer and isresponsible for transporting cells or particles from a prepared sample throughthe instrument for data acquision Fig. (3-2). The primary component of thissystem is a flow chamber. The fluidic design of the instrument and the flowchamber determine how the light from the illumination source ultimatelymeets and interrogates particles. Typically, a diluent, such as phosphatebuffered saline, is directed by air pressure into the flow chamber. This fluid isreferred to as sheath fluid and passes through the flow chamber after which itis intersected by the illumination source. Then, the sample under analysis, inthe form of a single particle suspension, is directed into the sheath fluid streamprior to sample interrogations. The sample then travels by laminar flowthrough the chamber (Ormerod, 1994).
  • 52Figure (3-1): Facscaliblur flow cytometry instrument.
  • 53Figure (3-2): Flow cytometer system (Facscalibur) (Ormerod, 1994). The pressure of the sheath fluid against the suspended particles alignsthe particles in a single file fashion. This process is called hydrodynamic
  • 54 focusing and allows each cell to be interrogated by the illuminationsource individually while traveling within the sheath fluid stream (Radcliffand Jaroszeski, 1998). The flow cell is the functional core of the fluidic system because itpresents cells in a single file for interrogation by the cytometer illuminationsystem. A typical flow cell Fig. (3-3) consists of a converging nozzle in whichsample is introduced at low flow rates into a larger laminar flow of isotonicsaline or sheath fluid. The cells in the sample follow the convergingstreamlines and are hydrodynamically focused into alignment. The sample isinjected into the center of a sheath flow. The combined flow is reduced indiameter, forcing the cell into the center of the stream. This the laser one cellat a time. This schematic of the flow chamber in relation to the laser beam inthe sensing area (Philip, 2002).1-2-Illumination system: Flow cytometers use laser beams that intersect a cell or particle thathas been hydro dynamically focused by the fluidic system. Light from theillumination source passes through a focusing apparatus before it intersects thesample stream. This apparatus is a lens assembly that focuses the laseremission into a beam with an elliptical cross-section that ensures a constantamount of particle illumination despite any minor positional variations ofparticles within the sample stream (Zimmermann and Truss, 1979).
  • 55 Laser optionsFigure (3-3): Flow cytometers use the principle of hydrodynamicsfocusing for presenting cells to a laser (Philip, 2002).
  • 56 Light and fluorescence are generated when the focused laser beamstrikes a particle within the sample stream. These light signals are thenquantitated by the optical and electronics system to yield data that is inter-prêtable by the user (Shapiro, 1995). Two systems are used in flow cytometry to focus the illuminatinglight to the point at which it intersects the cell stream. One type of system usesa spherical lens to give a focal spot size of 30- 60 µm. The second system usesa pair of crossed cylindrical lenses to focus the light to a sheet about 120 µmwide and 4-7 µm deep (Cledat et al., 2004). Most flow cytometers utilize a single laser, however, some systemssupport the simultaneous use of two or more different lasers. The mostcommonly used laser is an argon ion laser that has been configured to emitlight in the visible range of the spectrum. A 488- nm. Laser emission is usedfor most standard applications. The majority of fluorochrom that are availableon the market today can be excited using this wavelength. Thus, laser isexcellent excitation sources because they provide a single wavelength beamthat is also stable, bright, and narrow. Some type of lasers present in flowcytometers can be turned to U.V. or other wavelengths. If the exiting is nottunable, then another laser source that emits the desired wavelength isrequired. At the measuring point in a typical flow cytometer the stream of cellsintersects a beam of light from a laser or arc lamp Fig. (3-4). When lightinteracts with biological particles some of the light is scattered out of theincident beam and this scattered light may be collected over a range of anglesby detectors positioned around the measuring point (Gerstner et al., 2005).
  • 57Figure (3-4): A simplified illustration of Flow Cytometry (Gerstner et al.,2005).
  • 58 1-3-Optical and electronics system: The excitation optics consists of the light source and the opticalcomponents that serve to interrogate or excite the hydrodynamically focusedsample stream in the flow cell. The 488 nm line of the argon laser is used as alight source in many commercially available cytometers, but any light sourcethat provides the requisite intensity, e.g. , the mercury vapor or the xenon arelamp can be used. Optical components are used to expand, shape and focus thelight which then interacts with the sample in the flow cell. The flow cell isusually made of quartz and is designed to minimize diffraction and tomaximize the collection of the optical signals. The light source is often a laser.Laser is used because they provide a very concentrated and intense beam ofmonochromatic character of the light is especially important in makingfluorescence measurements (Telford, 2004). The amount of light that is scattered by a cell is a complex functionof its size, shape and refractive index. The sensitivity of light scattering to eachof these factors is dependent upon the range of angles over which the scatteredlight. The light scattered at small angles (i.e. forward light scatter) could besuccessfully used to determine relative volume distributions for populations ofcells (Wang et al., 2004). Light scattered and emitted in all directions (360º) after the laser beamstrikes an individual cell or particle that has been hydrodynamically focused.The optical and electronics system of a typical flow cytometer is responsiblefor collecting and quantitating at least five types of parameters from this scatterlight and emitted fluorescence. Two of these parameters are light scatteringproperties. Light that is scattered in the forward direction (in the samedirection as the laser beam) is analyzed as one parameter, and light scatteredat 90º relative to the incident beam is collected as a second parameter.
  • 59 Forward-scattered (FSC) light is a result of diffraction. Diffracted lightprovides basic morphological information such as relative cell size that isreferred to as forward angle light scatter (FSC). Light that is scattered at 90º tothe incident beam is the result of refracted and reflected light. This type oflight scatters is referred to as side-angle light scatter (SSC). This parameter isan indicator of granularity within the cytoplasm of cell as well as surfacemembrane irregularities to topographies (Philip, 2002). Most current laboratory bench-top flow cytometers are capable ofdetecting fluorescence from three different regions of the visible spectrum.Cutometers are optically conquered to detect a narrow range of wave lengthsin each region. Fluorescence emission is detected simultaneously along withFSC and SSC data; therefore, up to five parameters can be simultaneouslymeasured for each analyzed sample (Longobardi- Given, 1992) Fluorescence is detected using networks of mirrors, optics, and beamsplitters that direct the emitted fluorescent light toward highly specific opticalfilters. The filters collect light within the range of wave lengths associated witheach of the three fluorescent channels. Filtered light is directed toward photomultiplier tubes or PMTs for conversation into electrical signals (Telford,2004).1-4-Data storage and computer control system: After light scattering and fluorescence is converted to electricalsignals by the optical and electronics system, the information is converted intodigital data that the computer can interpret. The signals generated from cellsor particles are referred to as events and are stored by the computer (Rose etal., 1992).
  • 61 After the different signals or pulses are amplified they areprocessed by an Analog to Digital Converter (ADC) which in turn allows forevents to be plotted on a graphical scale (One Parameter, Two parameterHistograms). Flow cytometry data outputs are stored in the form of computerfiles (Radcliff and Jaroszeski, 1998). Histogram files can be in the form of one-parameter or two-parameterfiles. Histogram files consist of a list of the events corresponding to thegraphical display specified in your acquisition protocol. A one-parameterhistogram is a graph of cell count on the y-axis and the measurement parameteron x-axis. All one-parameter histograms have 1,024 channels. These channelscorrespond to the original voltage generated by a specific "light" eventdetected by the PMT detector. In other words, the ADC assigns a channelnumber based on the pulse height for individual events. Therefore, brighterspecific fluorescence events will yield a higher pulse height and thus a higherchannel number when displayed as a histogram. A graph representing twomeasurement parameters, on the x- and y-axes, and cell count height on adensity gradient. This is similar to a topographical map. You can select 64 or256 channels on each axis of two-parameter histograms. Particle counts areshown by dot density or by contour plot. Fig. (3-5) (Roederer et al., 2004). List-mode files consist of a complete listing of all eventscorresponding to all the parameters collected, as specified by your acquisitionProtocol. This file follows a format specified by the FCS 3.0 standard. Rawlist-mode data files can be opened or replayed using any program designed foranalysis of flow cytometry data. You should keep in mind that a Protocolserves as a template. It allows you to collect specified Parameters (i.e. FLS,FL1, FL2, etc.), and how these parameters are displayed. Protocols also serveto determine how the data is gated, and contains all the Regions from which
  • 60 your statistics will be generated. In addition, Protocols contain otherspecific information that serves as direct interface between the computerworkstation and the cytometer. These pertain to high voltage settings for thePMT detectors, gains for amplification of linear parameters, sample flow rates,fluorescence compensation, discrimination settings, etc. Once your data hasbeen collected and written into a list-mode file you can replay the file eitherusing the specific Protocol used for collection or any other programspecifically designed for analysis of flow cytometry data. However, youshould keep in mind that you can only adjust Regions, Gating, and Parametersto be displayed. Settings such as amplification, fluorescence compensation,etc., can not be modified. Therefore, when collecting data make sure that yourinstrument settings are correct. Finally, if you open your listmode files using aprograms such as FlowJo Fig. (3-6), WINMIDI, and/or ExPO you will haveto specify parameter displays, and create Regions and Gating correspondingto the Protocol used for collecting the data (McCoy, 2002).
  • 62Figure (3-5): Two parameter histogram and dot plot displaying FL1-FITC on the x axis and FL2-PE on the y axis (Roederer et al., 2004).
  • 63Figure (3-6): FlowJo program (McCoy, 2002).
  • 64 The number of events acquired for each sample is alwaysdetermined before analysis and is usually set using software designed tocontrol cytometer operation. A conventional acquisition value is 100.000events per sample. However this value may vary and range upward of eventsper sample depending on the experimental objective (Melamed et al., 1990).2-Data analysis: Data analysis is a very critical part of any experiment that utilizesflow cytometry. Data is analyzed using a computer and software is usuallyspecific to flow cytometric data and is often part of the same computer systemthat is used to control the instrument during acquision. The most commondisplay is a histogram. A typical histogram data plot is shown in Fig. (3-7, 3-8) (Abu- Absi et al., 2003). It is also possible to display two parameters simultaneously such asFSC vs SSc or FL1 vs FL2. For two parameter plots, data from a populationof individual particles can be displayed in the form of dots or as contoursshown in Fig. (3-9) (Parks and Henzenberg, 1989). Contour density plots display the data from a population of cells as aseries of concentric lines that correlate to different cell or particle densitieswithin the axes. Dot-plots are probably the most common type of two-parameter plots, and they are also the easiest to understand (Robinson, 1993).
  • 65Figure (3-7): Analysis pulse width versus pulse height or area we caneliminate the majority of G0 doublets that appear as G2 (Abu- Absi et al.,2003).
  • 66Figure (3-8): DNA histogram (Abu- Absi et al., 2003).
  • 67Figure (3-9): DNA histogram (aneuphliod population) (Parks andHenzenberg, 1989).
  • 68 IV- Applications of flow cytometry Flow cytometers are very complex instruments that are composed offour closely related systems. They provide a very unique and powerfulanalytical tool for researchers and clinicians. Therefore, the flow cytometer iswidely used in research as well as in clinical immunology and hematology toperform rapid immunophenotyping, cell sorting, and DNA analysis(Longobardi-Given, 1992 and Bogh & Duling, 2005). Flow cytometry is used for immunophenotyping and DNA content ofa variety of specimens including whole blood, bone marrow, serous cavityfluids, cerebrospinal fluid, urine and solid tissues. Characteristics that can bemeasured include cell size, cytoplasmic complexity, DNA or RNA content,and a wide range of membrane-bound and intracellular portents and sortingthe cells (Rechtnwald, 1993, Ormerod, 1994 and Assuncao et al., 2005). The use of flow cytometry in the clinical laboratory has grownsubstantially in the past decade. This is attributed in part to the developmentof smaller user friendly, less expensive instruments and a continuous increasein the number of clinical applications as shown in Table (4-1) (Brown andWittwer, 2000). Flow cytometry provides rapid analysis of multiple characteristics ofsingle cells. The information obtained is both qualitative and quantitative.Where as in the past flow cytometers were found only in larger academiccenters, advances in technology now make it possible for community hospitalsto use this methodology (Orfao et al., 1995 and McCoy, 2002).
  • 69 Table (4-1): Common clinical uses of flow cytometry (Brown andWittwer, 2000). Common characteristic Field Clinical application measures Immunology Histocompitability crossmatching IgM, IgG Transplantation rejection CD3, CIRCULATING OKT3 HLA-B27 detection HLA-B27 Immunodeficiency studies CD4, CD8 Oncology DNA content and S phase of tumors DNA Measurements of profilation markers Ki-67, PCNA Hematology Leukemia and Lymphoma phenotyping Leukocyte surface antigens Identification of prognostically important Tdt, MPO subgroups Hematopiotic progenit or cell enumeration CD34 Diagnosis of systematic mastocytosis CD25,CD69 Reticylocyte enumeration RNA Autoimmuneand alloimmune disorders Antiplatelats disorders IgG, IgM Anti-neutrophils antibodies IgG Immune complexes Complement, IgG Feto-maternal hemorrge quantification Hemoglobin F, rhesus D Immunohematology Erthrocyte surface antigens Assessment of leukocyte contamination of blood Forward AND Side scatter products Genetic PNH CD55,CD59 disorders
  • 71 Functions of cells can be defined through the application offluorochrome dyes that have an affinity for cellular components. Traditionally,common clinical applications are immunophenotyping of cells of thehematopoietic system with fluorescent-labeled antibodies raised againstspecific cell surface proteins (Davis et al., 2002). Other approaches have been used to elucidate changes in cell functionand DNA content. Examples of clinical applications in equine patients includeimmune-mediated hemolytic anemia, immune-mediated thrombocytopenia(IMT), chronic inflammatory disease, and neoplasia (Davis et al., 2002). The great advantage of flow cytometry is that its applications arehighly amenable to standardization. The efforts that have been made for flowcytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration,screening for the HLA-B27 antigen and leukemia/lymphomaimmunophenotyping (Keeney et al., 2004). The diagnosis of many primary immunodeficiency diseases requiresthe use of several laboratory tests. Flow cytometry is applicable in the initialworkup and subsequent management of several primary immunodeficiencydiseases (Illoh, 2004).1- Cell cycle analysis: The measurement of the DNA content of cells was one of the firstmajor applications of flow cytometry and is still one of the biggest applicationsin this laboratory today (Albro et al., 1993).
  • 70 Flow cytometry offers the possibility to study several parameterssimultaneously, e.g. cell cycle modulation, mode of cell death, activity ofmitochondria. The phases of the cell cycle were determined and the inductionof apoptosis and necrosis was demonstrated by annexin binding, analysis ofmitochondrial membrane potential and DNA strand breaks (Tusch andSchwab, 2005). DNA ploidy and proliferative activity (S-phase fraction) are the twobiological parameters commonly measured by DNA flow cytometric analysis.The prime purpose of most studies is the investigation of the prognostic valueof DNA flow cytometry in addition to the information provided byconventional clinicopathological factors known to affect disease prognosis.The general statement, for tumors in the same histopathological stage of thedisease, is that diploid and/or low proliferative tumors have a more favourableprognosis than aneuploid and/or high proliferative tumors, suggesting animportant role of DNA flow cytometry in the assessment of tumor behaviourand in the outcome evaluation of the disease. (Pinto et al., 2002).1-1-Staining procedure: The preparation and staining of cell suspension are the major factorsdetermining the validity and reproducibility of flow cytometric analysis. Thereis no flow cytometric staining procedures which is universally accepted and anumber of different protocols have been advocated. All of the DNA specificstains and the phenanthridinium dyes have been used for total DNA stainingof chromosomes. The former group has the potential disadvantage that UVexcitation is required but this constitutes no problem for mercury arc lampbased system or those with a laser tunable to UV lines (Hartwell, 1998).
  • 72 The DNA fluorochromes in current use were classified intogroups. Stains that intercalate with double stranded nucleic acid and include(PI), (EB) and (AO);and DNA specific stains that have a particular specificityfor moieties in DNA and include mithramiycin (MMC), ethediumbromide/mithramicin (EB/MMC), a bisbenzimidazole derivative and 4-6diamino-2- phenylindole (DAPI) (Taylor and Mithorpe, 1980). Propidium iodide is bound to polynucleotide both by means ofintercalation. This is only affected to a limited extent by high ionic strengthsand electrostatically to secondary binding sites. The binding contents of theselater sites are greatly depending on the ionic strength of the medium and canbe eliminated by using sufficiently high ionic strengths. And also have optimalexcitation at a 488 nm laser and produce good results with RNAse treatment(Hartwell, 1998).1-2-Evaluation of DNA histogram: The DNA histogram is a very simple data set which characteristicallycontains two peaks separated by a trough Fig.(4-1). The first peak, which is usually the larger, corresponds to cells withG0/G1 DNA content and the second, which should be at double thefluorescence intensity of the first, corresponds to cells with G2+M DNAcontent. Any cell scored in the trough has a DNA content intermediatebetween G1 and G2+M and these usually represent cells in S-phase. In aperfect data set, which doesnt exist, all G1 and G2+M cells would be scoredin single channels and any cells between or immediately adjacent to thesewould be in S- phase (Henderson, 1998).
  • 73 Since the cell material always contained normal diploid cells suchas leukocytes or normal kidney cells, these were used as an internal standardand regarded as diploid (2C) (Tribukati, 1984). When in doubt, lymphocytes should be added to establish the diploidDNA value. Human Ficol-prepared lymphocytes, fixed in ethanol, were usedas external standard. The magnitude of the signal was adjusted so as to havethe standard diploid peak in a certain channel. In necessary the illuminationwas adjusted so that the coefficient of variation (CV) of the resultinglymphocyte diploid peak was less than 3% Fig. (4-1). All cell population withG1 maximum deviating less than 10% form the standard value were regardedas diploid (Gustafson, 1982). Only G0/G1 peak is observed in DNA diploid. A broad peak describedby a large coefficient of variation may obscure a second peak. The coefficientof variation of the G0/G1 peak must be less than 5% for single cell suspensionsprepared from fresh/ frozen tissues, and less than 8% for nuclear suspensionprepared from fixed, paraffin embedded specimens. Where a diploid peak onlyis observed, one should ensure that tumor cells are present in the clinicalsample analyzed (Weinberg, 1996).
  • 74 Full width at half maximum(c-a) C.V. = ‫%5.24× ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Peak (or mean) channel(b)Figure (4-1): Coeffecient of Variation (C.V.) (Gustafson, 1982).
  • 75 An aneuploid cell population was considered to be present whena distinct peak was found constituting at least 2.5% of the total cell materialand deviating more than 10% from the diploid standard. DNA aneuploid isreported when at least two separate G0/G1 peaks are demonstrated. For somesamples the diploid normal peak might be almost non existent; hence careshould be taken to assign peaks (Ormerod, 1994). The degree of ploidy of this cell line was calculated by relating theG1 maximum of these cells to DNA of the diploid G1 cells which are alwayspresent. These diploid cells can be leukocytes, fibroblasts, normal urothelialcells. 2 channel of aneuploid peakDegree of ploidy= ———————————— Channel number of diploid peak The degree of aneuploid was determined also by the DNA indexwhich represents the ratio of fluorescence intensity of aneuploid cells to thediploid cells. The DNA index of a diploid tumor is 1.0, whereas, aneuploidtumors are designated by progressively higher indices (Götte, 2001 andGorden et al., 2003). Estimation of the proportion of G1, S and G2+M cells made byautomatic integration of the cells in corresponding channels in the multichannel analyzer. The values are corrected for background noise. To estimatethe proportions of cells in the S-phase of aneuploid tumor cell lines where cellsfrom the cell line coincide, the normal cell lines are subtracted. Calculation ofthe phase distribution requires a minimum of about 1000 cells combined witha low background noise resulting from cell fragments. These calculations mayalso be rendered more difficult when the peaks for the deploid or aneuploid
  • 76 cell population, partially or completely overlap. On the other hand, thereis no ambiguity in any of these cases to establish an aneuploid cell line. Anadditional proof of the existence of an aneuploid hyperdiploid cell line is theexistence of a G2 peak occurs to the extreme right of the histogram withoutinterfering with the normal cell population (Brown and Wittwer, 2000). An aneuploid cell population with a tetraploid amount of DNA wasconsidered to exist when a peak exceeded the G2+ M peak found in normalcells by three standard deviations or more. To quantitate the number of nucleinormally found in the 4C or G2/ M peak, a number of control tissues werestudied. The mean percentage of nuclei in the 4C peak were 2.74+ 1.41(standard deviation) for nuclei extracted from fresh normal lymphocyte. Thesecontrol data provide a firm basis for using greater than 10% of nuclei in the4C peak as a criterion for DNA polyploidy in the specimens (Rabinovitch,1994).2- Immunophenotyping Applications: The most common applications of flow cytometry are measurementof DNA content in tumors and immunophenotyping of haematopoieticmalignancies. Flow cytometry has shown to be a suitable method forimmunophenotyping of canine lymphomas (Culmsee and Nalte, 2002). Immunophenotyping of abnormal cells is now considered afundamental tool to establish the cell lineage assignment and to obtain a moreprecise identification of the various cell subtypes. Diagnostichematopathology depends on the applications of flow cytometricimmunophenotyping and immunohistochemical immunophenotypingcombined with the cytomorphology and histologic features of cases. Theavailability of monoclonal antibodies directed against the surface proteins
  • 77 permits flow cytometric analysis of erythrocytes, leukocytes and platelets(Brown & Wittwer, 2000, Chianese, 2002 and Dunphy, 2004). Multiparameter flow cytometry with optimally selected antibodycombinations has expanded the use of this technique beyond traditionalapplications in hematopathology. By analyzing qualitative patterns of antigenexpression on discrete populations or "clusters," one can detectimmunophenotypic aberrancy in specific cell populations relative to normaland reactive populations. Evaluation of patterns of antigen expression can alsobe used to supplement conventional methodologies in the diagnosis andsubclassification of certain types of hematologic neoplasia. Finally, thediagnosis of some congenital disorders affecting the hematolymphoid systemcan be facilitated by the detection of characteristic immunophenotypicchanges (Kroft, 2004).2-1-Erythrocyte analysis: Tests that appear to have the greatest potential for routine applicationof flow cytometry include reticulocyte and reticulated platelet enumeration,detection of erythrocyte-bound immunoglobulin, immunophenotyping ofleukemias and lymphomas, and bone marrow differential cell counting(Brown and Wittwer, 2000 , Weiss, 2002). Flow cytometric methods were first applied to laboratory hematologywith the improvement in reticulocyte counting and the creation of theimmature reticulocyte fraction for better anemia evaluation and therapeuticmonitoring (Davis, 2001).
  • 78 2-2-HIV monitoring: More than 35 million people in developing countries are living withHIV infection. While drug prices have dropped considerably, the cost andtechnical complexity of laboratory tests essential for the management of HIVdisease, such as CD4 cell counts, remain prohibitive. New, simple, andaffordable methods for measuring CD4 cells that can be implemented inresource-scarce settings are urgently needed (Dieye et al., 2005, Walker et al.,2005 and Pattanapanyasat & Thakar, 2005).2-3-Immunophenotyping of leukemias: Immunophenotyping has become common in the diagnosis andclassification of acute leukemias and is particularly important in the properidentification of cases of minimally differentiated acute myeloid leukemia. Toevaluate the immunophenotype of adult AML, cases were studied bycytochemical analysis and by flow cytometry with a panel of antibodies(Khalidi et al., 1998). Characterization of leukemias by immunotyping is particularly helpfulwhen the morphology is difficult to interpret. The major advantage of usingimmune markers by flow cytometry is the identification of particular leukemiasubtype, not recognized by morphologic criteria, which may have prognosticsignificance (Rezaei et al., 2003). Flow cytometric immunophenotypic analysis allowed to establishdiagnosis in cytomorphologically unclassified cases, identify acute mixed-lineage leukemias (AMLL) with a frequency similar to that reported in otherseries, and confirm the heterogeneity of acute leukemia (AL) (Piedras et al.,1997 and Götte, 2001).
  • 79 Flow cytometry may be used to detect minimal residual disease(MRD) in acute lymphoblastic leukemia because leukemic cells often displayaberrant phenotypes when compared to normal cells. Flow cytometry is asensitive and specific method for detecting MRD of childhood ALL, and couldpredict the coming relapse (Zhang et al., 2005). With the advent of monoclonal antibodies and a uniformnomenclature system defining antibody reactivity in terms of clusters ofdifferentiation (CD), an independent means of characterizing acute leukemiasusing cellular antigen expression has evolved. Immunophenotyping is usuallyperformed using immunofluoresence technique and is complementary to thelight microscopic based morphologic classification. This is especially true ofthe lymphoid leukemias where morphology and cytochemistry cannotdistinguish among different lineage of lymphoid cells, such as B versus T cells.With Immunophenotyping lineage is assigned using a panel of monoclonalantibodies that identify the expression of cell surface antigens. The panel ofmonoclonal reagents must include antibodies reactive with both myeloid andlymphoid cells to distinguish between the two most important groups. Thereactivity pattern of the leukemia cells for all reagents is then examined for thefinal assignment of lineage: B- lymphoid, T- lymphoid, myeloid orundifferentiated (Maslak et al., 1994). Comparative studies of cell surface antigen expression betweennormal and leukemic cells indicate that most if not all leukemias expressphenotypes that are not observed in most normal maturing cells. This aberrantexpression of cellular antigens suggests that leukemias are not proliferationsof cells arrested at one state of normal maturation; rather leukemic cellsmaintain a genetic program that can produce expression of antigens of any
  • 81 lineage. Nearly all laboratories performing immunofluoresence analysisuse different reagents (Terstappen et al., 1991).2-4-Quantification of stem cells: 100 years ago, hematopoietic stem cells were postulated as bloodlymphocyte-like cells. Within the last 20 years, the frequency of autologousand allogeneic transplantation of hematopoietic stem cells has increased.Hematopoietic growth factors allow the stem cells mobilization from the bonemarrow into the peripheral blood. Quantification of these hematopoietic stemcells by means of flow cytometry can be achieved within hours (Goldschmidtet al., 2003). Flow cytometry has become the major technique for the qualitycontrol of stem cell-containing products such as apheresis concentrates, bonemarrow or cord blood (Grieson et al., 1995). Stem cells can be easilyidentified with flow cytometry due to their unique characteristics. Theydemonstrate a medium level of CD34 expression, a low level of CD45expression and a low forward side scattered (Jennings & Foon, 1997 andMaslak et al., 1994).2-5-Platelet analysis: The analysis of platelets by flow cytometry is becoming morecommon in both research and clinical laboratories. Platelet-associatedimmunoglobulin assays by flow cytometry can be direct or indirect assays,similar to other platelet-associated immunoglobulin immunoassays. Inautoimmune thrombocytopenic purpura, free serum antibodies are not foundas frequently as platelet-bound antibodies (Ashman et al., 2003).
  • 80 Immunofluorescent flow cytometry was used to measure thepercentage of activated platelet populations (CD62P, CD63), the percentageof plt-monocyte aggregates (pma) (CD41/CD45), and activated monocytes(CD11b, CD14, CD16) in the blood (Panasuik et al., 2005).2-6-Testing for HLA-B27: Human leukocyte antigen B27 (HLA-B27) is a majorhistocompatibility complex class 1 molecule that is strongly associated withthe disease ankylosing spondylitis. The performance of the two flowcytometric antigen assays depends on the antibody used and the positive cutoffvalues assigned (Seipp et al., 2005). A flow cytometric HLA-B27 test is muchfaster than the classical microcytoxicity test (Jennings and Foon, 1997 andGötte, 2001). A biannual external quality assurance scheme for flow cytometrictyping of the HLA-B27 antigen is operational in The Netherlands and Belgiumsince 1995. For flow cytometry, the most widely monoclonal antibody usedwas FD705, followed by GS145.2 and ABC-m3. The majority of laboratoriesused more than 1 anti-HLA-B27 monoclonal antibody for typing (Seipp et al.,2005).3- Major applications of apoptosis analysis: There are many ways of detecting apoptosis by flow cytometry.Apoptotic cells can be recognized by a characteristic pattern of morphological(cell shrinkage, cell shape change, condensation of cytoplasm, nuclearenvelope changes, nuclear fragmentation, loss of cell surface structures,apoptotic bodies, cell detachment and phagoctosis of remains), biochemical
  • 82 and molecular changes (free calcium ion rise, bcl2/Bax interaction, celldehydration, loss of mitochondrial membrane potential, proteolysis,phosphatidylserine externalization, lamin B proteolysis, DNA denaturatuin,50-300kb cleavage, intranucleosomal cleavage and protein cross-linking(Hubank et al., 2004 and Liu et al., 2004). The methods of detecting apoptosis by flow cytometry are based onthe measurement of light scatter, the detection of changes in the plasmamembrane, the analysis of cell organelles or the sensitivity of DNA todenaturation (Sedlak et al., 1999).3-1-Apoptosis light scatter: As cells die or become apoptotic the refractive index of the internalcytoplasm becomes more similar to that of the extracellular medium thismanifests itself as a reduction in forward scatter signal. At the same time,intracellular changes and invagination of the cytoplasmic membrane lead to anincrease in side (or orthogonal or 90º) scatter. If a dead cell discriminatory dyeis added, cells that have become permeable can be identifying. In this waylow level resolution of dead and apoptotic cells can be get. A number of deadcell dyes are available for use and the one used will depend on any otherfluorochromes that are being measured. Some examples are; Sytox Green(488nm excitation; green fluorescence emission), Propidium Iodide (488nmexcitation; orange/red fluorescence emission), 7-Aminoactinomycin-D (7-AAD) (488nm excitation; red fluorescence emission) and TO-PRO-3 (633nmexcitation; red fluorescence emission) (Cohen and Al-Rubeai, 1995).
  • 83 3-2-Apoptosis DNA analysis: During apoptosis, calcium and magnesium dependent nucleases areactivated which degrade DNA. This means that within the DNA there are nicksand fragmentation. We can detect these in three ways using DNA analysis tolook at the sub G1 peak, using strand break labeling (TUNEL) to detect brokenDNA or using Hoechst binding to detect DNA conformational changes(Majino and Joris, 1995). The sub-G1 Fig. (4-2) method relies on the fact that after DNAfragmentation, there are small fragments of DNA that are able to be elutedfollowing washing in either PBS or a specific phosphate-citrate buffer. Thismeans that after staining with a quantitative DNA –binding dye, cells that havelost DNA will take up less stain and will appear to the left of the G1 peak. Theadvantage of this method is that it is very rapid and will detect cumulativeapoptosis and is applicable to all cell types (Darzynkiewicz, 1997). However in order to be seen in the sub G1 area, a cell must have lostenough DNA to appear there, so if cells enter apoptosis from the S or G2/Mphase of the cell cycle or if there is an aneuploid population undergoingapoptosis, they may not appear in the sub G1 peak (Schwartz and Osborne,1993).
  • 84Figure (4-2): Sub G1 peak by propidium iodide staining (Darzynkiewic,1997).
  • 85 Also cells that have lost DNA for any other reason e.g. death bysome other form of oncosis, will appear in the sub G1 region so we have to becareful about how we define the sub G1 peak (Nicoletti et al., 2001).3-3-Apoptosis cell membrane analysis: In normal cells, phosphatidylserine (PS) residues are found in theinner membrane of the cytoplasmic membrane. During apoptosis, the PSresidues are translocated in the membrane and are externalized. In generalthough not always, this is an early event in apoptosis and is though to be asignal to neighboring cells that a cell is ready to be phagocytosed (Robinson,1993). Annexin-V is a specific PS-binding protein that can be used to detectapoptotic cells. Annexin V- is available conjugated to a number of differentfluorochromes. Early apoptotic cells are annexin positive but PI negative.Because the cells arent fixed we can exclude dead cells and it is possible toadd further markers if the cytometer set up are appropriate. As with all livecell assays, we have to remember that we are only looking at a snapshot of thecells as they are at time of analysis and generally all apoptotic experimentsshould be performed over a time course; Fig. (4-3) (Telford et al., 2004,Homburg et al., 1995 and Vermes at al., 1995).
  • 86Figure (4-3): Early apoptotic cells are annexin positive but (in this case)PI (negative) (Telford et al., 2004).
  • 87 Hoechst 33342 is a DNA-binding dye that is able toquantitatively stain the DNA of live cells. However it has also been found thatif the concentration of Hoechst is low, the apoptotic cells take up the Hoechstmore rapidly. If we also add PI or TO-PRO-3 we can specifically identify thedead cells. This is a rapid and quantitative method but requires the use of aUV laser. The advantage of using TO-PRO-3 is that cell phenotyping usingFITC- and PE-labelled antibodies is also possible. Thymocytes labelled withCD4-PE and CD8-FITC can be assessed for apoptosis using Hoechst and TO-PRO-3 (Koopman et al., 1994). A third way of assessing the membrane changes in apoptosis is to useYO-PRO-1 (Molecular Probes). As this fluorochrome emits in the green, itcan be combined with propidium iodide to identify dead cells. The rationalehere is that cells in early apoptosis are unable to pump out YO-PRO-1 but arestill not permeable to other dead cells discriminatory dyes (Koopman et al.,1994).3-4-Apoptosis enzyme analysis: Two genes (ced-3 and ced-4) were crucial to the process ofapoptosis. The ced-4 gene product has homologues in mammalian cells,especially a family of cysteine proteases that are now known as caspases.There are a number of caspases in mammalian cells that have been shown tobe involved in the early stages of apoptosis e.g. (caspase2, caspase3, caspase6, caspase 9 and caspase 10). The functions of these enzymes are not yetentirely clear but it appears that after an initial signal to the cell to undergoapoptosis, they may be responsible for the activation, amplification andexecution of the apoptotic cascade (Cohen and Al- Rubeai, 1995).
  • 88 Because of the central importance of the caspases in apoptosis,their detection by flow cytometry has become widespread. We can detect theactivity of enzymes implicated in apoptosis in three ways; by detecting theactive form of the enzyme using a specific antibody (Smolewski et al., 2002),by using a fluorochrome labelled peptide that binds to the active site of theenzyme (Pozarowski et al., 2003) and by using a non-fluorescent substrate forthe enzyme which yields a fluorescent product if the enzyme is active (Telfordet al., 2004).3-5-Apoptosis organelle analysis: During apoptosis there is often a collapse of the mitochondrialmembrane potential. This can be detected in a number of ways by flowcytometry. Two dyes in particular are useful- CMXRos (also known as Mitotracker Red from Molecular probes) and LDs-751 (from Exction). CMXRoshas a chloromethyl group which allows accumulation in activemitochondria. Live cells that have active mitchondria are able to take upCMXRos but in cells that are undergoing apoptosis, the mitochondrialmembrane potential decreases which means less dye accumulates in themitchondria leading to a decrease in fluorescenc (Chapman et al., 1995).4- Detection of apoptotic markers: Determination of p53 expression by immunohistochemistry (IHC) hasbeen incorporated into routine practice and its reliability has beenconsolidated. However, flow cytometric (FCM) analysis might represent animportant objective and rapid approach. FCM may provide importantinformation about p53 protein expression in the different subpopulations andcell cycle phases. In most breast, lung, and colon aneuploid tumors (77%),
  • 89 p53-positive cells were detected only in the subpopulations withabnormal DNA content (Elvira et al., 1998). Bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblastcell lines were analyzed via flow cytometry (FCM) for expression of p53 andc-myc proteins along with their DNA content. At least 9,000-10,000 p53 or c-myc protein molecules per cell were detected in the transformed tumorigeniccell lines. These results show that quantitative FCM can be reliably used todetect very low levels (3,000 molecules per cell) of specific protein, and FCMis a useful tool to study the virus-induced changes in the levels of nuclearproteins within a cell population and in tumorigenesis (Agrawal et al., 1994). In human follicular lymphoma, Analysis of transgenic Bcl2expression used biotinylated Bcl2-100 monoclonal antibody for the surfacephenotyping of hematopoietic cells by flow cytometry. Cells (106 per analysis)were stained with relevant antibodies labeled with fluorochromes (fluoresceinisothiocyanate [FITC], phycoerythrin [PE], or cyanin 5 [Cy5]) or biotin using1% normal rat serum to block Fc receptors. Streptavidin conjugated to FITCor PE was used as a secondary reagent for biotinylated antibodies. Analyseswere performed on a Life Sciences Research (LSR) or a FACStar II flowcytometer (Becton Dickinson, San Jose, CA) (Alexander et al., 2004). The expression of bcl-2 was examined by multicolor flow cytometryin samples including lymph node or other tissue biopsy specimens containingfollicular lymphoma (FL), reactive hyperplasia (RH), or other malignantlymphomas, as well as bone marrow aspirates. For all of the aforementionedreasons, a reliable flow cytometric assay for expression of bcl-2 would be auseful additional technique for establishing a diagnosis of FL. However, the
  • 91 measurement of bcl-2 by flow cytometric techniques has received onlyscant attention. It was described a 2-color flow cytometric assay usingantibodies against bcl-2 that demonstrated promise in the recognition of FL.(James et al., 2003).
  • 90 V-Flourescence in situ hybridization1-Introduction: FISH provides researchers with a way to visualize and map thegenetic material in an individuals cells, including specific genes or portions ofgenes. This is important for understanding a variety of chromosomalabnormalities and other genetic mutations. Unlike most other techniques usedto study chromosomes, FISH does not have to be performed on cells that areactively dividing. This makes it a very versatile procedure. The first step is toprepare short sequences of single-stranded DNA that match a portion of thegene the researcher is looking for. These are called probes. The next step is tolabel these probes by attaching one of a number of colors of fluorescent dye(Schröck et al., 1996 and Fox et al., 1996). DNA is composed of two strands of complementary molecules thatbind to each other like chemical magnets. Since the researchers probes aresingle-stranded, they are able to bind to the complementary strand of DNA,wherever it may reside on a persons chromosomes. When a probe binds to achromosome, its fluorescent tag provides a way for researchers to see itslocation (White et al., 1995 and Bloom, 2005). Fluorescent in situ hybridization (FISH) represents a modemmolecular pathology technique, alternative to conventional cytogenetics(karyotyping). Fluorescence in situ hybridization (FISH) allows identificationof specific sequences in a structurally preserved cell, in metaphase orinterphase (Kontogeorgos, 2004 and Celedaet al., 1994).
  • 92 The probe, bound to the target, will be developed into afluorescent signal. The fact that the signal can be detected clearly, even whenfixed in interphase, improves the accuracy of the results, since in some casesit is extremely difficult to obtain mitotic samples Fig. (5-1) (Muhlmann,2002). The power of in situ hybridization can be greatly extended by thesimultaneous use of multiple fluorescent colors. Multicolor fluorescence insitu hybridization (FISH), in its simplest form, can be used to identify as manylabeled features as there are different fluorophores used in the hybridization.By using not only single colors, but also combinations of colors, many morelabeled features can be simultaneously detected in individual cells using digitalimaging microscopy (Raap et al., 1995). Fluorescence, a phenomenon whereby a chemical excited at one lightwavelength emits light at a different and usually longer wavelength, is usedthroughout the life sciences to study a wide variety of structures andintracellular activities. Advances in probe and microscope technology have ledto the rapid development of techniques for fluorescence over the past decade(Trask, 1991). The accuracy of cytogenetic diagnosis in the management ofhematological malignancies has improved significantly over the past 10 years.Fluorescence in situ hybridization (FISH), a technique of molecularcytogenetics, has played a pivotal role in the detection of unique sub-microscopic chromosomal rearrangements that helped in the identification ofchromosomal loci, which contain genes involved in leukemogenesis (Amareet al., 2001).
  • 93Figure (5-1): Fluoresence in situ hypridization (Muhlmann, 2002).
  • 94 The use of FISH is growing rapidly in genomics, cytogenetics,prenatal research, tumor biology, radiation labels, gene mapping, geneamplification, and basic biomedical research. In principle, the technique isquite straightforward (Attarbaschi et al., 2004). The hybridization reaction identifies, or labels, target genomicsequences so their location and size can be studied. DNA or RNA sequencesfrom appropriate, chromosome-specific probes are first labeled with reportermolecules, which are later identified through fluorescence microscopy. Thelabeled DNA or RNA probe is then hybridized to the metaphase chromosomesor interphase nuclei on a slide. After washing and signal amplification, thespecimen is screened for the reporter molecules by fluorescence microscopy(Hohman and Gundlach, 1994). FISH probes are commonly used to detect the presence of specificDNA sequences either when DNA is condensed into metaphase chromosomesor dispersed in non dividing interphase cells. The fact that hybridization ofprobes to metaphase chromosomes is visualized in two dimensions whileinterphase targets are three dimensional has implications for both validationsof assays and the development of baseline reference ranges (Pauletti et al.,1996). Metaphase applications generally yield clear yes/no answers whileinterphase applications commonly require reportable reference ranges beforeinterpreting of results. In addition to determining the presence or absence ofparticular sequences in the genome, FISH is useful in assessing gene copynumber in some disorders (Massod et al., 1998).
  • 95 Analytical uncertainty over DNA probe assays also may stemfrom issues related to inherent population variation. The use of some repeatsequence probes has been discontinued because of inability to detect targetedsequences in individuals who possess very few repeats, leading to insufficientprobe label in the targeted region which precludes visualized of the signal.Such probes have been eliminated (Myrata et al., 1997 and Bossuyt et al.,1995). FISH allows very precise spatial resolution of morphological andgenomic structures. The technique is rapid, simple to implement, and offersgreat probe stability. The genome of a particular species, entire chromosomes,chromosomal-specific regions, or single-copy unique sequences can beidentified, depending on the probes used (Attarbaschi et al., 2004). Until recently, FISH was limited by the hardware, software, reagents,probe technology, and cost involved in implementing the technique.Commercially available microscope hardware optimized for multicolor FISHwas not available until the mid-1990s. Prior to that, microscopes had to becustomized for FISH applications. Most microscope optics was not designedto detect the low light levels inherent in FISH signals. As the genomicresolution of the technique has increased dramatically, the requirements onmicroscope optics have further increased. Chromatic aberrations amongmultiple wavelengths have been a problem. For multicolor analysis inparticular, all lenses, including the collector lens, had to be chromaticallycorrected. In addition, epi-fluorescence light sources were difficult to align foruniform illumination (Amare et al., 2001). Analysis of multicolor FISH images requires isolation of the varioussignals either with individual filter cubes; or utilization of an excitation filter
  • 96 wheel with multipass dichroic and barrier filters. Recent developments infilter technology corrected some of the previous problems encounteredthrough optical misalignments caused by mechanical switching of individualfilter cubes. Excitation filter wheels used with multi-pass dichroic and barrierfilters can be used effectively for up to three colors by employing separateexcitation filters for each color with no registration shift. But, for more thanthree colors, single-pass filters still had to be used (Racevskis, 2005, Iarovaiaet al., 2005 and Iourov et al., 2005).2-Three different types of FISH probes:2-1-Locus specific probes They bind to a particular region of a chromosome. This type of probeis useful when scientists have isolated a small portion of a gene and want todetermine on which chromosome the gene is located (Hjalmar, 2005 andWang et al., 2005).2-2-Alphoid or centromeric repeat probes They are generated from repetitive sequences found in the middle ofeach chromosome. Researchers use these probes to determine whether anindividual has the correct number of chromosomes. These probes can also beused in combination with "locus specific probes" to determine whether anindividual is missing genetic material from a particular chromosome (Edwardet al., 2005).
  • 97 2-3-Whole chromosome probes They are actually collections of smaller probes, each of which bindsto a different sequence along the length of a given chromosome. Usingmultiple probes labeled with a mixture of different fluorescent dyes, scientistsare able to label each chromosome in its own unique color. The resulting full-color map of the chromosome is known as a spectral karyotype. Wholechromosome probes are particularly useful for examining chromosomalabnormalities, for example, when a piece of one chromosome is attached tothe end of another chromosome (Dugan et al., 2005).3-Applications of FISH: The clinical uses of FISH were considered in three main areas;diagnosis of individuals with birth defects and mental retardation, prenataldiagnosis and screening, and identification and monitoring of acquiredchromosome abnormalities in leukemia/ cancer. In each area the criticalconsideration remains a clear understanding of the capabilities and limitationsof a test to provide useful information (Bossuyt et al., 1995 and Pauletti et al.,1996). Traditional cytogenetic analysis, detecting deletions, duplications,rearrangement and the identifications of unknown material of marker orderivative chromosomes, in individuals with birth defects and/or mentalretardation has led to an understanding of the etiology of a number ofsyndromes. The clinical utility and limitations of these tests are both generaland disease specific (Ledbeteer et al., 1987, Callen et al., 1992, Ribeiro et al.,1997 and Cassidy et al., 1998).
  • 98 Prenatal applications of FISH testing include both screening testsand diagnostic tests. Technical issues are few, and clinical utility raisesquestions as to the intended use of FISH in testing. The application of FISH toprenatal screening for common autosomal trisomies and sex chromosomeanomalies is becoming increasingly common. The primary considerationsinvolve differing clinical sensitivity between the abnormalities detected byclassical cytogenetic versus these detected by FISH based assays (Evans et al.,1991 and Klinger et al., 1992). Among cases ascertained via ultrasonographically identified fetalanomalies, some may be conclusive for a syndromes diagnosis and may beapproached by a (diagnostic) FISH test. Families in which subtle orsubmicroscopic chromosomal abnormalities, detectable by FISH, are knownto segregate will benefit greatly from prenatal FISH studies (Kontogeorgos etal., 2000 and Lewin et al., 2000). Fluorescence in situ hybridization (FISH) has become one of themajor techniques in environmental microbiology. The original version of thistechnique often suffered from limited sensitivity due to low target copynumber or target inaccessibility (Zwirglmaier, 2005). The reagents and probes themselves were not sufficient for allapplications. For instance, the efficiency of hybridization site detectiondecreased with decreasing probe size, creating significant limits to what couldbe observed via fluorescence microscopy. The number of differently coloredfluorescent dyes was limited, and the photostability of the dyes was poor. Butnew developments in fluorescent dye technology and spin-off technology fromthe federally funded Human Genome Project are now having an impact. Thereare probes for all the human chromosomes and a growing number of new gene-
  • 99 specific probes are available. In situ hybridization kits and fluorescentlylabeled probes are commercially available from several companies (Sarrate etal., 2005). The ability of FISH to rapidly test interphase and metaphasechromosome defects makes it especially useful in the study of cancer. In solidtumors, conventional cytogenetics is rarely used because obtaining metaphasesis difficult and those cells that do proceed to mitosis may not be representativeof the tumor. Other molecular techniques, such as PCR and Southern,Northern, and Western analysis, require extraction of the tissue. Extractionprocedures net both normal and abnormal cells, so sensitivity is lower andquantitation less reliable than with FISH probes (Bosch et al., 2005). FISH allows cell-by-cell analysis and thus provides for a moresensitive and reliable assessment of chromosomal aneuploidy, geneamplifications and deletions, and chromosome translocations. A reliabledetermination of whether a gene is amplified in a specimen is often possiblewith evaluation of only 20 to 50 cells (Ogilvie et al., 2005). The accuracy of cytogenetic diagnosis in the management ofhematological malignancies has improved significantly over the past 10 years.FISH has played a pivotal role in the detection of unique sub-microscopicchromosomal rearrangements that helped in the identification of chromosomalloci, which contain genes involved in leukemogenesis (Amare et al., 2001). FISH was performed with specific probes to make the rapid prenataldiagnosis of Down syndrome. FISH was performed respectively with locus-specific probe (LSI) and centromeric probe (CEP) X/Y on the uncultured
  • 011 amniotic fluid. FISH is a rapid and reliable method to detect Downsyndrome in uncultured amniotic fluid (Wang et al., 2005) Fluorescence in situ hybridization assay and to correlate the geneticfindings with the pathologic grade and stage were used to investigate thechromosomal abnormalities present in bladder carcinoma (Placer et al., 2005). A novel application of FISH to isolated nuclei is described. Themethod detects gene amplification and chromosome aneuploidy in extractednuclei from paraffin-embedded tissue of human cancer with greater sensitivityand specificity than existing FISH methods. The method is applied to signaldetection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of themost common genetic alterations associated with human breast cancer (Rossiet al., 2005). Tumor-specific chromosomal abnormalities are attracting a largeinterest owing to the diagnostic, prognostic, and therapeutic importance. Thedevelopment of FISH has improved the detection of specific chromosomalabnormalities in chronic lymphocytic leukemic (CLL). By using FISH, theproblem with tumor cells with low mitotic rate is avoided since this methodreadily detects clonal aberrations also in nondividing, interphase cells. Threedifferent types of probes are used centromeric probes for numericalchromosome abnormalities, whole chromosome paints, and locus-specificprobes for numerical chromosome abnormalities, whole chromosome paints,and locus-specific probes. (Hjalmar, 2005) FISH of DNA-DNA or DNA-RNA using post-mortem brain samplesis one approach to study low-level chromosomal aneuploidy and selectiveexpression of specific genes in the brain of patients with neuropsychiatric
  • 010 diseases. FISH could be applied to extended studies of chromosomalaneuploidy, abnormal patterns of chromosomal organization and functionalgene expression in situ in the neurons of the brain in different psychiatric andneurodevelopmental diseases (Yurov et al, 2001).3-1-ALL investigation by FISH: To investigate patients with acute lymphoblastic leukemia (ALL) forTEL/AML1 fusion, BCR/ABL fusion, MLL gene rearrangements, andnumerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situhybridization (FISH) and to determine the relationship and the significance ofthose findings (Zhang et al., 2003). Interphase fluorescence in situ hybridization (iFISH) is increasinglyused for the identification of BCR/ABL gene rearrangements in chronicmyeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). FISHplays an important role in detecting chromosome changes, especially in somecryptic chromosome translocations and patients with culture failures (Primoet al., 2003 and Zhang et al., 2003). ALL blasts routinely contain somatically acquired geneticabnormalities that provide insight into pathogenesis and strongly influenceprognosis. Approximately one third of cases of ALL show an increase in themodal chromosome number (e.g., hyperdiploid, > 47 chromosomes, and"high" hyperdiploid, > 50 chromosomes) blasts make up a unique biologicsubset associated with increased in vitro apoptosis and sensitivity to a varietyof chemotherapeutic agents (Heerema et al., 2000 and Trueworthy et al.,1992).
  • 012 Almost one third of ALL blasts show chromosomaltranslocations in the absence of changes in chromosome number. Four majortranslocations have been observed, and each defines a unique biological subsetof patients. The t(1;19)(q23;q13) is a hallmark of some pre-B (cytoplasmic µ+)ALL, and is characterized by fusion of the E2A and PBX genes (Uckun et al.,1998). Despite the adverse prognostic impact of this translocation in olderstudies, recent intensification of therapy has resulted in an improved survivalfor these children. Translocations between the mixed lineage leukemia (MLL)gene at 11q23 and over 30 different partner chromosomes characterize 6% ofALL cases. MLL translocations, most commonly t(4;11)(q21;q23), are seen inthe vast majority of infant patients with ALL. A recent, large seriesdemonstrates that any rearrangement of 11q23 is associated with a worseprognosis (e.g., 20% to 25%) (Pui et al., 2002).3-1-1-Philadelphia The presence of the t(9;22)(q34;q11) translocation, commonly knownas Philadelphia chromosome (Ph), in about 3% to 5% of all children with ALLis considered as one of the molecular markers associated with a particularlyhigh risk for treatment failure (Ribeiro et al., 1987, Crist et al.,1990, Pui etal., 1990, Fletcher et al., 1991, Reiter et al., 1994, and Chessells et al., 1995). This translocation causes a rearrangement between the protooncogenec-ABL and a gene called the breakpoint cluster region (BCR). Whereas thebreaks in c-ABL occur mainly in the same region (between the exons a1 anda2) on chromosome 9, two different ones affect the breakpoint cluster regionon chromosome 22: the more frequent one (approximately in 2 of 3 of all
  • 013 cases) shows a break in the minor breakpoint cluster region (m-BCR)between the exons e1 and e2. This is predominant in ALL. In 1 of 3 of all Ph+ALL cases, the major (M-) BCR found between exons b2 and b3 or exons b3and b4 is affected. M-BCR is also found in nearly all patients with chronicmyelogenous leukemia (CML). Chimeric proteins of 210 kD (p210) and190 kD (p190) result from the M-BCR/ABL and m-BCR/ABLrearrangements, respectively (Kantarjian et al., 1991). These fusion proteins cause a deregulation of protein tyrosine kinaseactivity. Both forms of the chimeric gene (BCR/ABL) can be detected bypolymerase chain reaction (PCR) and fluorescent in situ hybridization.(Maurer et al., 1991, Dewald et al., 1993, Schlieben et al., 1996). Most patients with Philadelphia (Ph)-positive acute lymphoblasticleukemia (ALL) show evidence of secondary chromosome aberrations thatmay influence the course of disease and response to treatment. To betterunderstand how these secondary chromosomal aberrations occur and toinvestigate whether the p185/p190 BCR-ABL fusion protein may directlyinduce an increased chromosomal instability and subsequently the appearanceof clonal chromosome aberrations, three BRC-ABL (p185/ p190)-transducedmouse pre-B cell lines were analyzed by spectral karyotyping and fluorescencein situ hybridization. The human wild-type BCR-ABL gene was expressed ata level comparable with that in human Ph-positive leukemias at diagnosis. AllBCR-ABL-transduced cell lines acquired similar clonal chromosomalaberrations. Trisomy 5 was always present, followed by loss of the Ychromosome, trisomy of chromosomes 12 and 18, and an unbalancedtranslocation between chromosomes X and 12. Thus, ectopic p185/p190 BCR-ABL expression, such as p210 BCR-ABL, PML-RARA, or C-MYCtransduction, may induce an increased chromosomal instability leading to
  • 014 clonal karyotypic evolution, which may mimic secondary chromosomeaberrations in human Ph-positive ALL (Rudolph et al., 2005). The Philadelphia (Ph) chromosome, the main product of the(9;22)(q34;q11) translocation, is the cytogenetic hallmark of chronic myeloidleukemia (CML), a clonal myeloproliferative disorder of the hematopoieticstem cell; the Ph chromosome is also found in a sizeable portion of acutelymphoblastic leukemia (ALL) patients and in a small number of acutemyeloid leukemia (AML) cases. Three different breakpoint cluster regions arediscerned within the BCR gene on chromosome 22: M-bcr, m-bcr, and mu-bcr(Drexler et al., 1999). Nearly all Ph + ALL cell lines have the m-bcr e1-a2 fusion gene(only two ALL cell lines have a b3-a2 fusion) whereas all CML cell lines, butone carry the M-bcr b2-a2, b3-a2 or both hybrids. The mu-bcr e19-a2 has beendetected in one CML cell line. Four cell lines display a three-way translocationinvolving chromosomes 9, 22 and a third chromosome. Additional Phchromosomes (up to five) have been found in four Ph + ALL cell lines and in18 CML cell lines; though in some cell lines the extra Ph chromosome(s)might be caused by the polyploidy (tri- and tetraploidy) of the cells. Anothermodus to acquire additional copies of the BCR-ABL fusion gene is theformation of tandem repeats of the BCR-ABL hybrid as seen in CML cell lineK-562. Both mechanisms, selective multiplication of the der(22) chromosomeand tandem replication of the fusion gene BCR-ABL, presumably lead toenhanced levels of the fusion protein and its tyrosine kinase activity (geneticdosage effect). The availability of a panel of Ph + cell lines as highlyinformative leukemia models offers the unique opportunity to analyze thepathobiology of these malignancies and the role of the Ph chromosome inleukemogenesis (Drexler et al., 1999).
  • 015 Treated children with acute lymphoblastic leukemia wereanalysed for chromosomal abnormalities with conventional G-banding,spectral karyotyping (SKY) and interphase fluorescent in situ hybridisation(FISH) using probes to detect MLL, BCR/ABL, TEL/AML1 rearrangementsand INK4 locus deletions. Three novel TEL partner breakpoints on 1q41, 8q24and 21p12 were identified, and a recurrent translocation t(1;12)(p32;p13) wasfound. In addition, two cases displayed amplification (7-15 copies) of AML1.Results were demonstrated the usefulness of SKY and interphase FISH for theidentification of novel chromosome aberrations and cytogenetic abnormalitiesthat provide prognostically important information in childhood ALL(Nordgren et al., 2002). The BCR/ABL and MLL/AF4 fusion genes--resulting fromt(9;22)(q34;q11) and t(4;11)(q21;q23) translocations, respectively--areconsidered as a high risk prognostic factors in children with acutelymphoblastic leukaemia (ALL). Their presence in malignant cells indicatespatient for the most intensive antileukaemic therapy regardless of the othercriteria. In contrast, the most common non-random chromosomal aberration inpaediatric ALL--translocation t(12;21)(q12;q22)--is associated with afavourable prognosis. The examination of these rearrangements is importantfor the stratification of patients to the risk groups and also provides the mostsensitive and specific tool for minimal residual disease (MRD) follow-up(Trka et al., 1999 and Poplack, 1993). VI- References
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  • 044 VII- LIFE FLOWCYTOMETRIC FIGURES (A) (B) M2 M1Figure (1): Flow cytometric analysis of c-myc expression on mononuclearcells showing diagram (A) and dot plot (B) of positively stained cells inrelation to negative ones. (A) (B) R2 M2 M1 R1
  • 045 Figure (2): Flowcytometric analysis of p53 expression onmononuclear cells showing histogram (A) and dot plot (B) of positivelystained cells in relation to negative ones. Diploid: 100.00% Diploid: 100.00% Dip G0-G1: 93.93 % at 33.49 Dip G0-G1: 90.43 % at 35.31 Dip G2-M: 4.57 % at 61.69 Dip G2-M: 7.84 % at 65.30 Dip S: 1.50 % G2/G1: 1.84 Dip S: 1.73 % G2/G1: 1.85 Dip %CV: 2.84 Dip %CV: 3.17Figure (3): Histogram showing cell cycle parameters (diploid) using flowcytometer FACS caliber programmodfit.
  • 046Diploid: 85.25 % Diploid: 62.08 % Dip G0-G1: 100.00 % at 38.52 Dip G0-G1: 100.00 at33.10 Dip G2-M: 0.00 % at 77.05 Dip G2-M: 0.00 % at 66.21 Dip S: 0.00 % G2/G1: 2.00 Dip S: 0.00 % G2/G1:2.00 Dip %CV: 3.42 Dip %CV: 3.16Aneuploid 1: 14.75 % Aneuploid 1: 37.92 % Aneup G0-G1: 65.14 % at 69.75 Aneup G0-G1: 95.37 % at45.19 Aneup G2-M: 28.72 % at 104.73 Aneup G2-M: 1.76 % at 73.95 Aneup S: 6.13 % G2/G1: 1.50 Aneup S: 2.88 % G2/G1: 1.64 Aneup %CV: 3.31 Aneup %CV: 3.43 Aneup DI: 1.81 Aneup DI: 1.37Figure (4): Histogram showing cell cycle parameters diploid andaneuploid using flow cytometer FACS caliber program modfit.
  • 047 VIII- LIFE FISH PICTURESFigure A Figure BFigure C Figure D Figure (A,B,C and D): Each childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 9 and green signal which is the breakpoint cluster region (BCR) on chromosome 22 for children with Philadelphia negative acute lymphoblastic leukemia (Ph‾ ALL).
  • 048Figure E Figure F Figure (E and F): Each childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 9, green signal which is the breakpoint cluster region (BCR) on chromosome 22 and pale orange signal which is the fusion (BCR/ABL) for children with Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL).