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TO STUDY POLYMOPHISM IN HEAT SHOCK PROTEINS:
HSP 70-HOM, HSP 70-1 and HSP70-2 GENE AND
EXPRESSION IN CHRONIC MYELOID LEUKEMIA
PATIENTS
UNDER THE GUIDANCE OF
PROF.DAMAN SALUJA
DR.B.R. AMBEDKAR CENTRE FOR BIOMEDICAL RESEARCH
UNIVERSITY OF DELHI, DELHI-7
PROJECT SUBMITTED BY:
LATIKA LUTHRA
In partial fulfillment of the degree M.Sc. Biomedical Sciences

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CONTENTS
Serial
number.
Topic Page
number.
1. Acknowledgement 3
2. List of Tables 5
3. List of Figures 6
4. Introduction 7
5. Review of Literature 9
6. Objective 20
7. Methods and Materials used 21
8. Observation and results 33
9. Conclusion 42
10. References 43

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ACKNOWLEDGEMENTS
Many learned souls put me on right path and enlightened me with their knowledge and
experience. It is a privilege to express my due sense of gratitude to all those who guide me
throughout this investigative period.
I am indebted to my guide Prof. DAMAN SALUJA, professor, Dr. B.R. Ambedkar centre
for biomedical research, University of Delhi for timely guidance and consistent support
throughout the research. She has been patient despite certain unsuccessful experiments during
the course of my work. And for this reason, I am thankful to Prof. Daman Saluja for giving
me the opportunity to work in her lab.
I also want to thanks Mrs. Sunita Jaitely for her invaluable suggestions and consistent
support throughout the project. I am really thankful to her for sharing her knowledge,
experience and clinical samples and cDNA preparations. Inspite of her heavy commitments
at the college, she has been constantly helping me in carrying out the experiments and
helping me in trouble shooting them. Her invaluable suggestions and consistent support
throughout the project has really been responsible for finishing this project in time. My deep
gratitude is also to Prof. K. Natarajan, Director of A.C.B.R.
I owe my heartiest thanks to all those who helped me during the project –the lab fellows and
seniors Dr. Meenakshi that angelic figure with motherly nature for always being there,
sharing her ideas and giving invaluable support, always helping me when I am super panicky.
Dr. Mashook Ali with whom probably I have interacted less but he has always given me
golden advice and helped me a lot when my stress levels were reaching sky. Ms Renu, Ms
Deepali, Dr. Subash C Sonkar, Ms Alka, Ms Indu, Ms Ahona, Ms Kirti, Dr. Manoj and
lab staff Ms Kalpana and Ms Pooja. I am also grateful to my class fellows, Ms Priya and
Ms Deepika who helped me emotionally and waiting for me till late for my reactions in lab,
helping me shuttling between USIC and ACBR. Ms Geetika Dalal my sister from another
mother ,thanks for being a great support for my masters, a sweetheart who kept my days all

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sunshine and blossom, for giving her ear for listening all my woes and always giving
legitimate advice. My heartiest thanks to Arun, without whose support, love and care I
wouldn’t have got the strength to complete my project, thanks for standing as a pillar when I
felt weak and motivating me to propel with force. Last but not the least thanks to my parents
and brother for having faith in me, understanding the pressure and my interest in doing the
project, accepting my odd timings of coming back home, always supporting my decisions and
being their whenever I needed support.
Ms Latika Lutra

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LIST OF TABLES
Table number. Topic Page number.
1. Types of HSPs and their functions 11
2. Types of HSPs genes 13
3. PCR primer sequences for HSP70-HOM,1
AND 2 GENE
25
4. PCR conditions for HSP70-1, HSP70-2 and
HSP70-HOM
28
5. Restriction enzyme used for different HSP
genes
29
6. Restriction digestion results interpretation 37
7. RFLP result of hsp70-hom 38
8. RFLP results of HSP70-1 39
9. RFLP result of HSP70-2 40

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LIST OF FIGURES
Figure number. Name of the figure Page number.
1. HSP and apoptosis 8
2. Function and synthesis of HSPs 10
3. Protein structure Hsp70 11
4. HSPs and their anti immune properties 12
5. Location of Hsp70-hom gene 14
6. Location of Hsp70-1 gene 14
7. Location of Hsp70-2 gene 15
8. Signaling pathways of p210BCR/ABL 17
9. Mechanism of action of Imatinib 18
10. Nanodrop spectrophotometer 23
11. Agarose gel electrophoresis 24
12. PCR thermal cycler 26
13. PCR reaction diagram 27
14. Image of gel doc system 28
15. Temperature gradient for HSP70-HOM 34
16. PCR product HSP70-HOM 34
17. RFLP Products for HSP70-HOM 38
18. PCR product HSP70-1 35
19. RFLP Products for HSP70-1 39
20. PCR product HSP70-2 36
21. RFLP Products for HSP70-2 40
22. Scatter plot of HSP70-1 41

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INTRODUCTION
The bcr-abl oncogene generated by the translocation (9;22) is one of the most highly studied
oncogenes. It is regarded as the diagnostic for chronic myeloid leukemia (CML). The
constitutive kinase activity of the Bcr-Abl fusion gene leads, via a myriad of signal
transduction pathways, to enhanced proliferation, genomic instability, and cytoskeletal
abnormalities and to decreased apoptosis. Although several molecular mechanisms resulting
in the antiapoptosis properties of Bcr-Abl have been unraveled, there are still many gaps in
our knowledge. Guo and colleagues attempted to elucidate the role of heat shock protein 70
(hsp70) in Bcr-Abl–mediated antiapoptotic pathways in human leukemic cells. It was shown
that Bcr-Abl expression (ectopic or endogenous) in leukemic cells results in the up-regulation
of the antiapoptotic hsp70 leading to induction and activation of signal transducers and
activators of transcription 5 (STAT5) and interference with several apoptosis signaling
pathways at multiple levels. Attenuation of HSP 70 levels either by imatinib or HSP70
antisense sensitized K562 cells to cytarabine (Ara-C), etoposide, and also Apo-2 ligand (Apo-
2L)/tumor necrosis factor (TNF)–related apoptosis-inducing ligand (Apo-2L/TRAIL)–
induced apoptosis. Higher levels of phosphorylated heat shock factor-1 (pHSF-1) were found
in Bcr-Abl positive cell lines, suggesting a regulatory role for HSF-1 in HSP70 expression.
Imatinib down-regulated pHSF-1 and HSP70 expression in Bcr-Abl –positive leukemic cells.
From these elegantly performed studies, the overall important conclusion can be drawn that
hsp70 plays a central role in both the extrinsic and intrinsic apoptotic signaling of bcr-abl–
positive leukemic cells, STAT5 dependent and independent. Most of the results were
obtained in cell lines; just overexpression of hsp70 and hsp90 was shown in primary cells
derived from blast crisis patient.

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Fig1.BCR/ABL tyrosine kinase activity increases Hsp levels which in turn blocks apoptosis
by silencing apoptosome formation and activating anti apoptotic pathways (Gert
Ossenkoppele, Blood, 2002)
The successful treatment of CML with the “Bcr-Abl–specific” tyrosine kinase inhibitor
imatinib leading to complete cytogenetic remissions in a high percentage of patients if
applied in chronic phase has transformed this disease into a paradigm for targeted therapy.
However, primary or secondary resistance is not an infrequent event and although complete
remissions can also be achieved in advanced stage CML.Therefore, the search for other
drugs, effectively targeting perturbed signal transduction pathways involved in the
pathogenesis of CML, is warranted. Switching off not only Bcr-Abl but also other key
proteins could potentially overcome primary as well as secondary resistance. Various new
small molecules are currently being tested either in vitro or in vivo. Hsp70 could be a suitable
target for these and, it is hoped, become the Achilles heel of the leukemic cell.

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REVIEW OF LITERATURE
HEAT SHOCK PROTEINS
Heat-shock proteins (HSPs) are molecular chaperones synthesized under stressful conditions.
They are important in physiological and pathological processes. HSPs are constitutively
expressed at low levels under physiological conditions whereas their levels increase in
cellular stress. They heal injured tissues by maintaining proper conformation of misfolded or
injured proteins. Their expression was triggered when organisms are exposed to a variety of
stressful stimuli including hypoxia, ischemia, and oxidative free radicals. Earlier it was
thought that HSPs are only present in cytoplasm and nucleus however now their role in
extracellular environment is seen in intercellular signaling. Family members of HSPs are
HSP90, HSP60, HSP70, HSP47 and HSP110, which are numbered based on their molecular
masses. It is reported that polymorphisms in the heat shock protein genes may contribute to
differential disease vulnerability because these proteins are involved in stress tolerance.
HSP70 is the most abundant chaperone protein in cells. It is quintessential for cell survival
under stress conditions. The human HSP70 family consists of 3 main genes: HSP70-1,
HSP70-2 and HSP70-HOM. HSP70-HOM encodes a non-heat inducible protein that shares
high homology with HSP70-1 and HSP70-2. These genes are polymorphic. The aim of this
study was to investigate the functional single nucleotide polymorphisms (SNP) of the HSP70-
HOM,HSP70-1 and HSP70-2 gene and their expression in chronic myeloid leukaemia.

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Fig.2 The pathophysiological signals that activate Hsp synthesis (Benjamin, Circ Res, 1998)
SINGLE NUCLEOTIDE POLYMORPHISM
Single-nucleotide polymorphism (SNP) is characterized by a variation in DNA sequence
which is detectable when a nucleotide differs in a biological species or pair of an individuals
chromosomes. Recombination, natural selection and mutation rate determines SNP density.
Polymorphisms are depicted in disease susceptibility in different individuals and how each
responds to a particular therapy. For example, a single base difference in the Apo lipoprotein
E is associated with a higher risk for Alzheimer's disease.

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Table 1. Different HSPs with their functions (Kapoor et al.,2013, AJMDS)
Fig.3. Protein structure of Hsp70 (Protein Data Bank)

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Fig.4. Anti immune properties of heat shock proteins. Tumor cells (pale blue), cancer cells
with a cuboid epithelial shape and spindle-shaped cancer stem cells (CSCs) suggesting the
EMT (epithelial mesenchymal transition) characteristics accredited to CSC. The tumor is
represented as a heterogeneous cell colony containing myeloid suppressor cells (MDSCs;
green), Treg (dark blue), and tumor-associated fibroblast. (Kapoor et al, 2013, AJMDS)
IL-10 and TGFβ are dominant cytokines in the tumor microenvironment. Growth factors like
FGF and VEGF are secreted by TAF. In the left of the image above are CD4+ T cells (red)
that have stalled at the tumor capillary wall. It has been shown that tumor cells are secreting
Hsp70-containing exosomes (black circles) that recruit MDSCs and free Hsp70 can also
trigger immunosuppressive responses. In the lower image it is suggests that the potential
effects of therapy using molecular chaperone vaccines are secretion of IL-6 at high levels
making the cytokine profile proinflammatory, CTL has crossed the capillary wall and
penetrated the tumor interstitial spaces where they recognized MHC class I associated with
tumor antigens. Hsp70 peptide complexes (Hsp70.PC) are also secreted from necrotic tumor
cells and can trigger anticancer CTL after entering antigen presenting cells and their cross-
presentation to CD4+ T cells in afferent lymph nodes.

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TABLE 2. FAMILY OF HSP70
Approved
Symbol
Approved Name
Previous
Symbols
Synonyms Chromosome
HSPA1A heat shock 70kDa protein 1A HSPA1 HSP70-1 6p21.3
HSPA1B heat shock 70kDa protein 1B HSP70-2 6p21.3
HSPA1L heat shock 70kDa protein 1-like HSP70-HOM,hum70t 6p21.3
HSPA2 heat shock 70kDa protein 2 14q23
HSPA4 heat shock 70kDa protein 4 HS24/P52, HSPH2 5q31.1
HSPA4L heat shock 70kDa protein 4-like APG-1, Osp94, HSPH34q28
HSPA5 heat shock 70kDa protein 5
(glucose-regulated protein,
78kDa)
GRP78 BiP 9q33.3
(HSP70B')
1q23.3
(HSP70B)
1q23.3
HSPA8 heat shock 70kDa protein 8 HSPA10 HSC71, HSC70,
HSP73
11q24.1
(mortalin)
HSPA9B GRP75, PBP74,mot-2,
mthsp75
5q31.1
HSPA12A heat shock 70kDa protein 12A FLJ13874, KIAA0417 10q25.3
HSPA12B heat shock 70kD protein 12B C20orf60 dJ1009E24.2 20p13
HSPA13 heat shock protein 70kDa family,
member 13
STCH 21q11.1
HSPA14 heat shock 70kDa protein 14 HSP70-4, HSP70L1 10p13
HSPH1 heat shock 105kDa/110kDa
protein 1
HSP105B, KIAA0201,
HSP105A, NY-CO-25
13q12.2-q13.3
HYOU1 hypoxia up-regulated 1 ORP150, HSP12A,
Grp170
11q23.1-q23.3

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HSP70-HOM
Official name : HSPA1L
Official full name: Heat shock protein 1-like
Gene type : protein coding
Organism : Homo sapiens
Location : 6p21.3
Sequence : Chromosome: 6; NC_000006.11 (31777396..31782835, complement)
Fig 5.Hsp70-hom gene (adapted from gene card database)
The gene is located in the major histocompatibility complex class III region, in a cluster with
two closely related genes which also encode isoforms of the 70kDa heat shock protein.
HSP70-1
Official name : HSPA1A
Official full name: Heat shock protein family A (Hsp70) member 1A
Location : 6p21.3
Sequence : Chromosome: 6; NC_000006.11 (31777396.31782835, complement)
Fig.6. Hsp70-1 gene (gene card database)

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HSP70-2
Official name : HSPA1B
Official full name: Heat shock protein family A (Hsp70) member 1B
Location : 6p21.3
Sequence : Chromosome: 6; NC_000006.11 (31777396..31782835, complement)
Synonyms : HSP70-2; HSP70.2; HSP70-1B
HSP70-2 is an intronless gene encoding a 70kDa heat shock protein. The gene is
located in the major histocompatibility complex class III region, in a cluster with two
closely related genes ie. HSP70-HOM and HSP70-1which encode similar proteins.
Fig.7.Location Hsp70-2 gene (gene card database)
Leukaemia is cancer of the blood cells. Most blood cells form in the bone marrow. In
leukemia, cancerous blood cells form and crowd out the healthy blood cells in the bone
marrow.
Classification
General classification
Leukemia is subdivided into a variety of large groups. The first division is between its acute
and chronic forms:
 Acute leukemia is rapid increase in the number of immature blood cells.

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 Chronic leukemia is excessive buildup of relatively mature but abnormal white blood
cells. Taking months or years to progress.
According to the kind of blood cell affected leukemias are divided into lymphoblastic
or lymphocytic leukemias and myeloid or myelogenous leukemias:
 In lymphoblastic or lymphocytic leukemias,the lymphoid branch of blood cells are
affected.
 In myeloid or myelogenous leukemias, the cancerous changes in myeloid proginator
cells giving rise to red blood cells, some other types of white cells, and platelets.
Chronic myelogenous leukemia
Chronic myeloid leukemia (CML) is cancer of the white blood cells. Unregulated growth of
primarily myeloid cells in the bone marrow and the accumulation of these cells in the blood
are the key characteristics of CML. Proliferation of mature granulocytes (neutrophils,
eosinophils and basophils) and their precursors is evident in CML. A characteristic
chromosomal translocation called the Philadelphia chromosome is hallmark of CML. CML is
mainly treated with targeted drugs called tyrosine kinase inhibitors (TKIs).This has led to
improved long-term survival rates.
Classification
Based on clinical characteristics CML is divided into three phases. In the absence of
treatment, CML begins in the chronic phase. After several years this progresses to an
accelerated phase and finally to a blast crisis which behaves like acute leukemia. Drug
treatment will usually stop this progression if started early. Acquisition of new chromosomal
abnormalities (in addition to the Philadelphia chromosome) is responsible for these phase
transitions.
Molecular pathophysiology of CML
Generation of a (9;22)(q34;q11) reciprocal chromosomal translocation in a hematopoietic
stem cell is the prime genetic event in CML. The translocation between the long arms of
chromosome 9 and 22 results in a shortened chromosome 22 which is known as the
Philadelphia chromosome (Ph) and it is found in over 90% of CML patients (Nowell and
Hungerford, 1960). A chimeric gene BCR/ABL on chromosome 22 (Rowley, 1973) and a
reciprocal ABL/BCR on chromosome 9 is found after the translocation as a molecular event.

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The ABL/BCR gene is transcriptionally active but it does not have any functional role in
CML and no ABL/BCR protein is formed. (Diamond et al., 1995; Melo et al., 1993)
The BCR/ABL protein encodes for a constitutively expressed tyrosine kinase which resultd in
phosphorylation of many cellular substrates and autophosphorylation of itself which in turn
induces recruitment and binding of a number of adaptor molecules (GRB2, CBL, SHC, and
CRKL ) and proteins. P210BCR/ABL leads to malignant transformation by activation of a
number of signal pathways interfering with basic cellular processes like control of cell
proliferation and differentiation. (Afar et al., 1994; Jiang et al., 2000; Puil et al., 1994;
Sawyers, 1993) adhesion (Bhatia et al., 1999; Gordon et al., 1987) and cell survival (Bedi et
al., 1994; Cortez et al.,1995; Cotter, 1995; McGahon et al., 1994)
Fig8. Signaling pathways of p210BCR/ABL (Salesse et al, Oncogene, 2002)
BCR-ABL Tyrosine Kinase Inhibitors
Imatinib Mesylate (STI571; Gleevec)
The BCR-ABL protein is considered an ideal target for imatinib, since the BCR-ABL
mutation is present in 95% patients with CML.

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Fig9.Mechanism of action of imatinib (Zaharieva et al.,2013)
HEAT SHOCK PROTEINS AND CANCER RISK
Distinct immunologic mechanisms affecting tumor cells and promoting cell growth has been
proposed to be associated to HSP70 expression (Wang et al., 2005 and Luk et al., 2006). In
cancer cells, the expression of HSP70 is abnormally high and the protein may participate in
oncogenesis and in resistance to chemotherapy (Garrido et al., 2006). Several single
nucleotide polymorphisms (SNPs) have been described in these genes. The most studied
regions are located at positions +1267 of HSP70-2 (rs1061581), +2437 of HSP70-hom
(rs2227956), and +190 of HSP70-1 (rs1043618). These SNPs could affect HSP70 expression
or function and further contribute to disease susceptibility and stress tolerance (Favatier et
al., 1997)
Several studies have investigated the association between the three HSP70 polymorphisms
and risk of cancer till now (Chouchane et al., 1997 and Wang et al., 2010). But still the
results remain controversial. Therefore, this study on polymorphism in CML pateints vs
healthy control is done to prove the association between CML and HSP 70 polymorphism
which can be used as a biomarker for future reference or a prognostic marker.
HSP 70 is the most abundant chaperone protein in the cell. The three main polymorphisms in
HSP 70 genes have shown association with many cancers, such as gastric, colorectal,
hepatocellular, and breast cancer. HSP70-2 (A+1267G) polymorphism located at the coding
region and is likely to affect the secondary structure of mRNA, thus affecting the stability of
mRNA and protein expression. HSP70-hom T+493C polymorphism has nonsynonymous
mutations, which leads to a Met to Thr substitution at position 493 in the peptide binding

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domain and may affect substrate binding specificity and chaperone activity of HSP70 (Milner
et al., 1992) . Some studies suggest that the frequency of the variant genotype was
significantly higher in patients as compared to controls in case of HSP70-2 anf HSP70- hom
polymorphism (Chouchane et al., 1997 and Ferrer et al., 2013). HSP70-1 G+190C
polymorphism base pair lies upstream of the translation initial site (Milner et al., 1992) . In a
study from Mexico, Partida-Rodríguez et al., 2010 reported that HSP70-1 C/G showed
significant association with gastric cancer.

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OBJECTIVE
 To study the genotypic pattern of HSP70-HOM, HSP70-1 and HSP70-2 gene
polymorphism.
 To study whether there is altered expression of these genes in CML patients.

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MATERIALS AND METHODS
WORK PLAN:
 Collection of blood sample from normal healthy individuals and CML patients.
 Genomic DNA isolation from control and patient samples.
 Genotyping in the study subjects by PCR-RFLP techniques.
 Interpretation of data obtained
 RNA Isolation
 cDNA preparation
 Quantitative RT-PCR to check for the expression.
CHEMICALS
Tris Base, EDTA, Agarose, Trizole Reagent and DEPC were purchased from Sigma
Chemicals Co. (USA). Ammonium chloride, Ammonium bicarbonate Pottasium dihydrogen
phosphate, disodium hydrogen phosphate and Ethidium Bromide were purchased from
Qualigens fine chemicals Pvt. Ltd. (USA). Sodium Chloride, Potassium chloride,
Chloroform, Iso-propanol, Ethanol, Glacial acetic acid and methanol were purchased from
Merck (USA). Taq Polymerase, dNTPs, Magnesium chloride, loading dye were purchased
from Bangalore Genei India Pvt Ltd. (India). SYBR Green mix was purchased from Rosche
(Germany).
STUDY SUBJECTS:
The healthy control study subjects are the volunteers from Delhi and surrounding area with
comparable socioeconomic-cultural background .These study subjects were residing within
the North India region for past three generation. In our study total 30 healthy controls and 30
patients with chronic myeloid leukemia were recruited from the outpatient department of
Haematology , All India Institute of Medical Sciences, New Delhi, India. Patients were given
all the information regarding the study and consent was obtained as per guidelines from
institutional ethical committee. The study did not influence the treatment of the patients.

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SAMPLE COLLECTION:
Five millilitre (5ml) of peripheral blood was collected from all the participants in a tube
containing 200µl of 0.5% ethylene diamine acetic acid (EDTA). The samples were divided in
two tubes, 3ml of whole blood was used for total RNA Isolation and remaining volume of
blood sample was used for the plasma separation at 2000rpm for 10mins.The pack cell
volume was used for the purpose of genomic DNA isolation.
GENOMIC DNA ISOLATION
Genomic DNA was extracted from whole blood leukocytes using the NUCLEOPORE DNA
MINI KIT as per the instructions given in the kit. Briefly, it is described as follows:
Blood (200µl)
↓
Mix with 200µl lysis buffer and 25µl proteinase K
↓
Vortex and put at 70oC for 1 hr, vortex in between
↓
Add 210µl ethanol after 1hr
↓
Load all in column and spin at 11000g for 1 min
↓
Throw supernatant
↓
Add 500µl buffer GBW (wash buffer)
↓
Centrifuge at 11000g for 1 min
↓
Discard supernatant
↓
Add 600µl buffer GB5(wash buffer)
↓
Centrifuge at 11000g for 1 min
↓

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Discard supernatant
↓
Dry spin at 11000g for 1 min
↓
Discard supernatant
↓
Put elution buffer at 700C
↓
Add 100µl of elution buffer (TE) in column
↓
Put at RT for 5 min
↓
Spin at 11000g for 1 min
↓
Collect supernatant
↓
Whole blood DNA collected
DNA QUANTIFICATION
DNA quantification was done by Nanodrop (Nanodrop spectrophotometer ND1000) which
measured DNA concentration in 1-2 µl samples. . Absorbance A260 and A280 was taken and
concentration of nucleic acids was calculated.
Figure 10. Nanodrop spectrophotometer (Google images)

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AGAROSE GEL ELECTOPHORESIS
The isolated DNA sample was checked by 1.5% Agarose gel electrophoresis. Agarose gel
electrophoresis was carried out in a horizontal matrix of agarose with 1x TAE buffer as
described by Sambrook et al.(1989). The required amount of agarose (depending upon the
percentage) was melted in conical flask containing 1x TAE. An aliquot of ethidium bromide
(10mg/ml) cooled to about 650C was added to get a final concentration of 0.5µg/ml. The
agarose solution was then poured on to gel tray sealed and fitted with comb. The agarose was
allowed to polymerize for 30 minutes. The comb was then removed gently and the agarose gel
(1.5%) was immersed in 1x TAE buffer in horizontal electrophoresis tank. The isolated DNA
samples were mixed with 1/6 volume of 6xloading buffer and electrophoresed at 5V/cm. A
100 bp DNA ladder was run in parallel as size standard. The bands were visualized using short
wave (300 nm) using transilluminator and photographed with a gel documentation system
Fig.11. Agarose gel electrophoresis to fractionate the DNA. Isolated DNA is mixed with
tracking dye and is loaded into agarose gel. The ethidium bromide intercalates with DNA and
enables us to see the DNA under UV light.
CRITERIA FOR DESIGNING PRIMER:
 The length of primers should be between 17-24 bases.
 Composition of base should be 50-60% (G+C).
 Primers should end(3’) in a G or C, or CG or GC:
This prevents “breathing” of ends and increases efficiency of priming.
 Preferred Tm should be between 55-800C.

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 3’ end of primers should not be complementary (i.e., base pair), as otherwise primer
dimers will be synthesized to any other product.
 Self complementarity of primer (ie. the ability to form 20 structures such as hairpins)
should be avoided.
 If three or more Cs or Gs at the 3’-ends of primers are present they may promote
mispriming at G or C – rich sequences (because of stability of annealing), and should
be avoided.
 Oligonucleotides
 All gene-specific primers were got synthesized from Biolinkk (USA) or Sigma
(USA). Molecular beacon was got synthesized from Sigma-Aldrich (USA). All
oligonucleotides are listed in Table 2.
TABLE.3. The details of primers used are shown below:
GENE SEQUENCE OF FORWARD
PRIMER
SEQUENCE OF REVERSE
PRIMER
HSP70-
HOM
5’- CAAGTCTGAGAAGGT
ACAGGA-3’
5’- GGTAACTTAGATT
CAGGTCTG-3’
HSP70-1 5’-ACCTTGCCGTGTT
GGAACA-3’
5’- GGTCTCCGTGACG
ACTTATA-3’
HSP70-2 5’-ATCTGCGTCTGCT
TGGTG-3’
5’- ATCAACGACGGAG
ACAAG-3’
POLYMERASE CHAIN REACTION:
Polymerase chain reaction (PCR) is a molecular biology technique to amplify a single or a
few copies of a piece of DNA many folds in magnitude thus generating thousands of copies
of a particular DNA sequence. It is based on phenomenon of thermal cycling which consists

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of cycles of repeated heating and cooling of the reaction for DNA. Denaturation, annealing,
extension and renaturation of DNA. Primers, DNA polymerase (Taq polymerase), dNTPs, buffer
and water are added in the reaction mixture which enables selective and repeated
amplification. As PCR progresses, the newly generated DNA itself becomes a template for
replication and is exponentially amplified after each cycle.
Fig.12. PCR thermal cycler (Google images)
The isolated DNA was subjected to PCR amplification by using Eppendorf Master Cycler.
The coding sequence of HSP70-HOM, HSP70-1 and HSP70-2 gene were amplified from the
genomic DNA, using sequence specific primers (forward and reverses primers) which were
designed with the help of the gene runner version 3.05 and obtained from Sigma-Aldrich,
(USA).
For amplification of gene of interest through PCR reaction was prepared containing all
ingredients as described below:
PCR COCKTAIL MIXTURE CONTAINS:
100ng of genomic DNA,
1µl of 10pmol of each primer( sense and antisense),
2.5µl of 2mmol dNTP,
2.5µl 10x Taq PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM
MgCl2),
0.5µl Taq polymerase in a final volume of 25µl.

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Fig. 13. Diagrammatic view of working of PCR(Google images)
Using Gradient PCR to Determine the Optimum Annealing Temperature:
Gradient PCR is a technique by which we can identify optimal annealing temperature which
uses the least number of steps. This optimization is generally achieved in just one experiment.
The Eppendorf Mastercycler Gradient provides a gradient function through which in one
single run we can evaluate up to 12 different annealing, elongation, or denaturation
temperatures.
Gradient PCR was used in the experiment in order to determine the optimal annealing
temperature of primer. Using the gradient function of the universal block, a gradient of 58-
62°C was set. The following test parameters were selected based on the results:

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PCR
CONDITIONS/GENES
HSP70-1 HSP70-2 HSP70-HOM
INITIAL HEATING
940C FOR 5 MIN 940C FOR 5 MIN 940C FOR 5 MIN
DENATURATION
940C FOR 30 SEC 940C FOR 45 SEC 940C FOR 45 SEC
ANNEALING
EXTENSION
REPEAT CYCLES REPEAT STEP 2 TO 4
FOR 35 CYCLES
REPEAT STEP 2 TO 4
FOR 35 CYCLES
REPEAT STEP 2 TO 4
FOR 35 CYCLES
FINAL EXTENSION
720C FOR 10 MIN 720C FOR 10 MIN 720C FOR 10 MIN
TABLE 4. PCR CONDITIONS FOR HSP70-1, HSP70-2 AND HSP70-HOM
RESOLUTION OF AMPLIFIED PRODUCT
The PCR products were electrophoresed on a 1.5% (HSP70-1) and 1% (HSP70-2 and
HOM) Agarose gel and detected with 3 µl Etbr to confirm the correct amplicon size. The size
of the amplified product as given in Table 3 was :
337bp for HSP70-1,
998bp for HSP70-2 and
878bp for HSP70-HOM
The bands were visualized by gel doc system.
Fig. 14. Image of Gel doc system(Google images)

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RESTRICTION FRAGMENT LENGTH POLYMORPHISM
PCR products of different genes were digested with different restriction enzymes as given in
Table 3. . The digested DNA was fractionated on agarose gel and ethidium bromide stained
bands were visualized using gel documentation system. The size of the digested fragments
were determined using the 100 bp plus DNA ladder.
GENES Size of the
amplicons
RESTRICTION
ENZYMES
%GEL FOR
VIEWING
RFLP
RESULTS
HSP70-1 337 bp BsrB1 2.5%
HSP70-2 998 bp Pst1 1.5%
HSP70-HOM 878 bp NCO1 1.5%
TABLE 5. Restriction enzyme used for the digestion of the amplicons for different Hsp.
Based on the size, the amplicons were fractionated on agarose gel of desired concentrations.
Protocol for digestion of amplicons with restriction enzymes directly after
amplification:
 PCR reaction mixture- 15µl (0.1-0.5µg of DNA)
 Nuclease free water- 6.5µl
 10X RE Buffer- 2.5µl
 Restriction enzyme- 1-2µl
 Mix gently and spin down for a few seconds.
 Incubate at 370C for 2 hr

30
RNA ISOLATION
Total RNA was extracted from whole blood leukocytes using the manual method as
described below:
Take 2-3ml of blood
↓
Spin at 5000rpm for 6min
↓
Discard serum
↓
Dissolve cells in 15ml lysis buffer(100ml-25mlof 1.4N NH4Cl and 2.5ml 1M NHCO3)
↓
Keep it for 35-40 min
↓
Centrifuge at 14000rpm for 15 min
↓
If clean pellet is observed (white pellet)
↓
Add 15ml 1X PBS
↓
Dissolve and centrifuge at 500rpm for 6min
↓
To the pellet add 750 µl to 1 ml trizol
↓
Separate aqueous phase
↓
Add 200-300 µl chloroform to aqueous phase
↓
Keep it for 15min at 40C
↓
Centrifuge at 14000g for 15-18min at 40C
↓
Decant

31
↓
Add 500µl chilled propanol to pellet
↓
Keep for half an hour at 40C
↓
Centrifuge at 14000g for 15-18min at 40C and decant
↓
Add 70% chilled ethanol
↓
Centrifuge at 9000g for 7-9 min at 40C and decant
↓
Dissolve pellet in RNase free water (20µl)
↓
Store RNA at -800C
The quality of RNA was checked by 2% agarose gel electrophoresis and quantified by using
Nanodrop (ND-1000).The yield of total RNA was in the range of 800-1000 nanogram/µl,
from which 500ng of total RNA was used for cDNA synthesis using First Strand cDNA
Synthesis kit (Fermentas) according to the manufacturer’s protocol as follows:
cDNA PREPARATION USING FIRST STRAND cDNA SYNTHESIS KIT
Total RNA (500ng) along with nuclease free water (upto 10µl) and 1ul random primer was
taken and incubated at 700C for 5 min,after which it was quick chilled on ice.the other
components such as 5X reaction buffer (4ul), Ribolock Rnase Inhibitor(1 l) and 10mM
dNTP mix(2µl)were added to the reaction mixture and incubated at 370C for 5 min. On
further steps, M-Mul V reverse trancriptase (2µl) was added to the reaction mixture and
incubated at 370C for 1 hr and then reaction was terminated at 700C for 5 min. The obtained
cDNA was quantified using Nanodrop spectrophotometer and then stored at -200C for qRT-
PCR.
QUANTITATIVE REAL TIME PCR
An Applied Biosystems Quant 6 flex Real time machine was used for quantitative real time
PCRs in this project. The system encompasses a heating block for thermal cycling and

32
detectors to measure fluorescence in each well of a 96-well optical plate. Fluorescence is
measured after every amplification cycle to quantify the accumulation of PCR product;
during the exponential phase of PCR cycling, the rate of product accumulation is proportional
to template concentration; relative template abundance and therefore be quantified by
monitoring increasing fluorescence in each well during temperature cycling. Real time
quantitative PCR was performed using Applied Biosystems Quant 6 flex Real time machine
with SYBR Green PCR Core reagents. Amplification was performed in triplicates in 15µl
volume, including 1µl cDNA, 1 pmol of each primer in 2X SYBR green PCR master mix
(Eurogentec). To amplify human transcripts for the patient of CML genes, we have used the
gene specific primer pairs. Real time PCR for all candidate genes was performed in triplicate
for each sample according to the protocol as follows:
After an initial holding step of 2 min at 500C and 10 min at 950C, samples were cycled 40
times at 950C for 15 seconds and 620C for 1 min. The melting curve analysis for amplified
product was used to confirm the specificity of real time PCR assay. Difference between the
CT values (delta –CT value) obtained for the gene of interest and normaliser or housekeeping
gene was calculated. The cycle threshold (CT) is defined as index of number of cycles
required for the fluorescent signal to cross the threshold whereas delta –CT value is the
difference between the CT values obtained for the gene of interest and normaliser or
housekeeping gene. The expression of all candidate genes was compared in controls and
patients group in terms of fold difference determined by delta-delta-CT equation:
delta-CT control = [CT value of gene of interest in control-CT value
of housekeeping gene in control]
delta-CT patient = [CT value of gene of interest in patient-CT value
of housekeeping gene in patient]

33
RESULTS AND DISCUSSION
Heat shock proteins (HSPs) are molecular chaperones that are important to maintain cellular
homeostasis by helping protein folding. Several family members have been shown to be
overexpressed in cells exposed to stress. A number of studies have revealed that HSP-70
and its isoforms are upregulated after exposure to oxidative stress, infections, under
inflammation as well as other pathological conditions. Hsp proteins have recently been
shown to be aberrantly expressed in a number of cancer cells including leukemia. Three
isoforms of Hsp70 have been mapped and reported. These are HSP70-1, HSP70-2, and
HSP70-HOM, of which HSP70-2 encodes the major heat-inducible HSP70. Although all the
three isoforms show polymorphism, the polymorphism at position 1267 of hsp70-2 results in
A to G transition at PstI site in the coding region. Individuals homozygous for the HSP70-2
G allele exhibit decreased expression of inducible HSP70-2 mRNA expression (Guo F 2005;
Steiner et al 2006). The altered expression in Hsp70 has also been shown to be associated in
increased drug resistance in AML and CML. All these results prompted us to look into
polymorphism in all the three genes of Hsp70, namely Hsp70-1, Hsp70-2 and Hsp 70.hom as
well as to look into the expression of these genes in imatinib treated patients and compare
them with age matched healthy subjects.
To begin with we standardized the conditions of PCR to amplify region spanning the
potential polymorphic site. Genomic DNA isolated from human samples were used for PCR
amplification using different annealing temperatures. Although a clear band was observed at
various temperatures, it is evident from Fgure 1 that the best annealing temperature were
found to be 600C for Hsp70-HOM. PCR was carried out using genomic DNA isolated from
thirty healthy volunteers and 30 CML patients. A representative figure using two random
genomic DNA samples from healthy volunteers and CML patients is presented as Figure 2
showing clear single amplicons under standard conditions at 600C for Hsp70-HOM.

34
Figure.15. Agarose gel showing amplicons obtained using different annealing temperature
and primers specific for Hsp70-HOM.
Figurge 16. Representative agarose gel(1%) electrophoresis pattern of PCR of HSP70-
HOM gene using genomic DNA isolated from samples .. PCR was carried out under
optimized conditions using specific primers and genomic DNA isolated from representative
samples where C1,C2 are healthy controls and P1 ,P2 are CML patients

35
We next standardized the annealing temperature for Hsp70-1 in a manner similar to the above
and observed that optimum temperature for annealing of primers under standard conditions
was also 580C to 600C and we observed a clear single band when gDNA isolated from
controls or patient samples was used as template.
Fig.17. Agarose gel (1.5%) electrophoresis showing amplicons for Hsp70-1. PCR for
HSP70-1 gene was carried out using gDNA template isolated from healthy controls (C1,C2)
and patient samples (P1).
.
Similarly, using different annealing temperatures, PCR for Hsp70-2 was standardized to get a
single band using genomic DNA of healthy controls as well as CML patients ( Figure 4). Our
results suggest that the optimum annealing temperature for the three genes is 60°C for HSP
70-HOM, while 62°Cwas found to be best for HSP70-1and HSP70-2. All subsequent
experiments were carried out using the respective annealing temperature.

36
Fig.18. Agarose gel(1%) electrophoresis pattern of PCR of HSP70-2 gene. PCR was
carried out under standarized conditions using genomic DNA isolated from healthy controls
(C1,C2) and CML patients (p1 and P2)..Restriction fragment length polymorphism( RFLP)
is a technique that can be used to check variations in homologous DNA sequences. In RFLP
analysis, the DNA sample is digested into pieces by restriction enzymes for a particular site
in gene and the resulting restriction fragments are separated according to their lengths by gel
electrophoresis. RFLP analysis was the first DNA profiling technique other than now used
DNA sequencing. Due to high cost and time associated with DNA sequencing, RFLP is
much wider used technique. In addition to genetic fingerprinting, RFLP was an important
tool in localization of genes for genetic disorders, genome mapping, determination of risk for
a disease and paternity testing. However, RFLP can only be performed if the polymorphism
or the mutation involves change in a restriction enzyme cleavage site. Fortunately, changes
we were looking for in the present study could be easily checked due to altered restriction
digestion in the two alleles (Table 6).

37
GENES RESTRICTION
ENZYMES
HOMOZYG
OUS WILD
TYPE
HOMOZYGOUS
(RARE)
HETEROZYGOUS
HSP70-1 BsrB1 GG
(337bp)
CC
(232 &105bp)
GC (337,232,105bp)
HSP70-2 Pst1 AA
(998bp)
GG
(770&228bp)
AG
(998,770,228bp)
HSP70-
HOM
NCO1 TT
(878bp)
CC
(551&327bp)
TC
(878,551,327bp)
Table 6: Representing the polymorphism and the fragments obtained/expected after
digestion with the respective restriction enzyme.
The amplicons obtained using genomic DNA and primers for Hsp70 -HOM amplified 878bp
band that can be cleaved by NcoI to check for the transition of T to C. ( Figure 5 and Table
1). This gave us two fragments of 551 and 327 bp if both the alleles were changed
(homozygous) while in heterozygous conditions we obtained three band (uncut 878bp band,
and cleaved DNA fragments of 551bp and 327 bp). We looked into the polymorphism of
Hsp70-hom in 30 controls and 30CML patients samples. A representative figure showing
polymorphism in controls and patient samples is shown in Figure 19. All patients were on
imatinib drug for more than one year. As evident from Table 6. we did not observe a
significant difference in the polymorphism in healthy controls and patient samples. It is also
possible that we need to expand our studies on larger number to really predict the association
of polymorphism with the disease if any.

38
Fig .19. A representative agarose gel (1.5%) showing pattern of RFLP of amplified
region of Hsp70-HOM after restriction digestion with Nco1 enzyme. Genomic DNA
isolated from healthy controls (C1 and C2) as well as patient P1 show three bands while that
from patient P2 shows only two band.
Table 7. Genotypic frequency of variant C2437T of HSP70-HOM gene in control and
patient subjects:
Genotype CONTROLS PATIENTS
TT 0 2
CC 28 25
TC 2 3
We next checked the polymorphism in Hsp70-1 and Hsp70-2 . The amplified product
obtained using specific primers for the two genes were digested with BsrB1 for Hsp70-1
while for amplicons obtained for Hsp70-2 we used pst1 enzyme. As shown in Figure 20 ,
BsrB1 digestion resulted in three fragments if it was heterozygous for G ( GC allele) while
two bands were observed in case of homogygous CC alleles and a single undigested band for
GG allele, As is evident from Table 6. Heterozygous alleles were most frequent in the

39
population although the rare CC allele was more frequent in patient samples. To get
statistically significant data, we need to extend our data on larger number of samples.
Fig .20.Agarose gel (2.5%) electrophoresis showing representative sample showing gene
polymorphism. Isolated gDNA was used as template for PCR for amplification with Hsp70-
1 and the amplicons were digested with BsrB1 enzyme.
Table 8. Genotypic frequency of variant G190C of HSP70-1 gene in control and
patient subjects:
GG 3 5
CC 2 6
GC 25 19
The HSP70-2 A+1267G polymorphism is a synonymous mutation located at the coding
region and is likely to affect the secondary structure of mRNA, thus affecting the stability of
mRNA and protein expression. We therefore also looked at the polymorphism in these
samples for HSP70-2. As evident from the figure, digestion of amplicons with pst1 enzyme
would lead to three fragments of 998bp ,770bp, and 228bp in heterozygous condition (AG)
while only two fragments will be visible in homozygous subjects (Table 6 and Figure 20).

40
As reported earlier, we also observed polymorphism in the population. However, unlike the
reports from western population, we did not see any association of polymorphism with the
patient samples. One of the obvious reason is the genetic variation due to ethinicity.
Figure 21. .Agarose gel (1.5%) electrophoresis pattern of RFLP of amplified gene
Table 9. Genotypic frequency of variant A1267G of HSP70-2 gene in control and
patient subjects:
AA 0 0
GG 2 2
AG 28 28
Individuals with AG/GG has been reported to be at a higher risk of developing cancer than
AA genotype carriers in Asians, Africans (Lei He et al.. 2014) , With regard to HSP70-hom
and HSP70-1 polymorphisms, although the sample size was small, we did not observe much
difference in the genotype distribution between CML patients and control. This observation

41
is inconsistent with earlier observation [M. Ferrer-Ferrer, 2013]. To check if instead of
polymorphism, the expression of Hsp70-1 may be different in healthy controls compared to
CML patients, expression of HSP70-1 gene at mRNA levels was determined by quantitative
real time PCR. The quantitative relative HSP70-1 gene expression with respect to the
expression of housekeeping gene,was determined by the delta CT method .All samples
including patients and controls group were executed in triplicates for HSP 70-1 gene as well
as for house keeping gene. The average delta CT value accounts for the relative gene
expression between patients and controls group and fold difference was calculated. The
average delta CT values of patients is 24.88.The scatter plot clearly depicts that the
expression of Hsp70-1 in patients was lower as compared to healthy controls. The altered
expression in Hsp70 has also been shown to be associated in increased drug resistance in
AML and CML. Since all these patients are under drug treatment it is evident from our
results that none of these patients are exhibiting drug resistance. Experiments with a larger
number of population is required to conclusively prove the hypothesis.
Figure 22. Scatter plot of gene expression of Hsp70-1 to compare relative expression in
controland patient samples. The delta Ct value was plotted .The X-axis represent the
patients and Y axis represents the delta Ct value.
0
5
10
15
20
25
30
0 5 10 15 20
scatter plot of
patients
-15
-10
-5
0
5
10
15
20
0 10 20 30 40
scatter plot of
controls

42
Conclusion
There is no significant change in the polymorphism patterns of patients and controls being
observed in the above samples in all three genes ie. HSP 70-HOM,HSP70-1 and HSP70-2
.The reasons could be
1)Less sample size
2)May be Hsp 70 is not a specific prognostic marker in CML
This analysis has limitations that must be acknowledged. First, because of incomplete raw
data or publication limitations, some relevant studies could not be included in our analysis.
Second, the controls included in our analysis were selected randomly either from north Indian
populations or various hospitals. Therefore, misclassification bias was possible because this
study might have included control groups who have different risks of developing cancer.
Thirdly, our results were based on unadjusted estimates, while lacking of the information
(such as age, gender, family history and other risk factors) for the date analysis may cause
serious confounding bias.
In summary, this analysis suggested that HSP 70-2, HSP70-hom and HSP70-1
polymorphisms was not associated with the risk of CML in Indian population. However,
large and well-designed studies taking into consideration gene-gene and gene-environment
interactions are warranted to validate our findings.

43
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