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Cancer P53 By Swati Seervi
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Cancer P53 By Swati Seervi

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a library assignment by swati seervi,B.Pharm final year Jodhpur pharmacy college,jodhpur,rajasthan.

a library assignment by swati seervi,B.Pharm final year Jodhpur pharmacy college,jodhpur,rajasthan.

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  • hi very nice reviews pls mail me for assingment plz my mailid dr,vetrivelpath@gmail.com
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    I found your post titled (cancer and p53) very interesting. I am grateful if you will send me the same on my id dpraveen2_bhu@yahoo.co.in.
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  • hello.today i have seen your post 'cancer p53', and actually I found this post is very useful which could help me about my work .I will be grateful to you if you can send this post to me . Thanks for your support .my email is 'wangqk 98738@163.com '.I am look forward to your email .
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    Cancer P53 By Swati Seervi Cancer P53 By Swati Seervi Document Transcript

    • Generated by Unregistered Batch DOC & DOCX Converter 2010.2.312.1374, please register! CANCER [p53 - the guardian angel of genome] [Abnormalities in the genes that regulate the cell death or apoptosis are associated with many diseases. For example, damage to genes called tumor suppressor genes, which produce proteins that normally inhibit cell division, causes some types of cancer. Loss or alterations of a tumor – suppressor genes called p53 on chromosome 17 is the most common genetic change.] Ms. Swati Seervi Bachelor of Pharmacy (Final Year) Jodhpur Pharmacy College Guide Mr.Sanjay Sharma
    • Cancer p53 jodhpur pharmacy collage 1. INTRODUCTION 1.1 A Short history about cancer Cancer has not been a new disease. The written descriptions about cancer can be found on Egyptian papyrus dating back to roughly 1600 B.C. The Egyptians blamed this disease on the gods and treated it with a cauterizing tool; they called it ―the fire drill‖. Then, apparently the drill did not happen to work, as the corresponding writing on the papyrus says ―there is no treatment‖ The Greek physician Hippocrates is believed to be the first person to use the word ―carcinos‖. Over the time this word became the now known ―cancer‖. When the first autopsy was performed by Italian anatomist Giovanni Morgagni in 1771, the foundation had been laid for the scientific study of cancer, thus the branch dealing with the study of cancer is called oncology. 1.2 A brief introduction about cancer Cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancer differentiate them from benign tumors, which are self-limited, and do not invade or metastasize. Most cancers form tumor but some, like leukemia, do not. Cancer affects people at all ages with the risk for most types increasing with age. Cancer caused about 13% of all human deaths in 2007 (7.6 million).cancer is caused by abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens, such as tobacco smoke, radiation chemicals, or infectious agents. Other cancer promoting genetic abnormalities may randomly occur through errors in DNA replication, or are inherited, and present in all cells by birth. Genetic abnormalities found in cancer typically affect two general classes of genes. Cancer – promoting oncogenes are typically activated in cancer cells, giving those cells new Page 1 of 62
    • Cancer p53 jodhpur pharmacy collage properties, such as hyperactive growth and division, protection against programmed cell death, loss of respect for normal tissue boundaries and ability to become established in diverse tissue environments. Tumor suppressor genes are then activated in cancer cells, resulting in the loss of normal functions in those cells, such as accurate DNA replication, control over the cell cycle, orientation and adhesion within tissues, and interaction with protective cells of the immune system. 1.3 Introduction to various types of cancers Cancer has been the most dreadful disease encompassing the world today. There are many types of cancer existing in present following are its types. 1.3.1 Types of ovarian cancers - Germ cell tumors ,  Rare tumors;  Sex cord tumors;  Epithelial tumors 1.3.2 Types of breast cancers - Ductal carcinoma  Lobular carcinoma;  Inflammatory breast cancer;  Medullary carcinoma of the breast;  Colloid carcinoma;  Papillary carcinoma 1.3.3 Metaplastic carcinoma of breast  Triple Negative Breast Cancer 1.4 Introduction about a tumor suppressor gene A tumor suppressor gene, or anti-Oncogene, is a gene that protects a cell from one step on the path to cancer. When this gene is mutated to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes. 1.4.1 Two-hit hypothesis Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis', which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the Page 2 of 62
    • Cancer p53 jodhpur pharmacy collage gene is damaged, the second can still produce the correct protein. In other words, mutant tumor suppressor‘s alleles are usually recessive whereas mutant Oncogene alleles are typically dominant. The two-hit hypothesis was first proposed by A.G. Knudson for cases of retinoblastoma.[1] Knudson observed that the age of onset of retinoblastoma followed 2nd order kinetics, implying that two independent genetic events were necessary. He recognized that this was consistent with a recessive mutation involving a single gene, but requiring biallelic mutation. Oncogene mutations, in contrast, generally involve a single allele because they are gain of function mutations. There are notable exceptions to the 'two-hit' rule for tumor suppressors, such as certain mutations in the p53 gene product. p53 mutations can function as a 'dominant negative', meaning that a mutated p53 protein can prevent the function of normal protein from the un-mutated allele. Other tumor-suppressor genes that are exceptions to the 'two-hit' rule are those which exhibit haploid sufficiency. An example of this is the p27Kip1 cell-cycle inhibitor, in which mutation of a single allele causes increased carcinogen susceptibility. 1.5 p53 as a tumor suppressor protein A protein that is the product of a tumor suppressor gene, regulates cell growth and proliferation, and prevents unrestrained cell division after chromosomal damage, as from ultraviolet or ionizing radiation. The absence of p53 as a result of a gene mutation increases the risk of developing various cancers. A 53 kD nuclear phosphoprotein encoded by the proto-oncogene p53, on chromosome 17p13; in its wild form, p53 inhibits cell growth control and transformation; it activates transcription of genes that suppress cell proliferation, acting as a tumor suppressor protein; if p53 is physically lost or functionally inactive, cells can grow without restraint. Page 3 of 62
    • Cancer p53 jodhpur pharmacy collage 2. HISTORY 2.1 The history of the tumor suppressor protein 53 p53 was identified in 1979 by Arnold Levine, David Lane and William Old, working at Princeton University, Dundee University (UK) and Sloan-Kettering Memorial Hospital, respectively. It had been hypothesized to exist before as the target of the SV40 virus, a strain that induced development of tumors. Although it was initially presumed to be an Oncogene, its character as a tumor suppressor gene was revealed in 1989.In 1993, p53 protein has been voted molecule of the year by the Science magazine. [12] p53, also known as TP53 or tumor protein (EC: 2.7.1.37) is a gene that codes for a protein that regulates the cell cycle and hence functions as a tumor suppression. It is very important for cells in multi cellular organisms to suppress cancer. P53 has been described as "the guardian of the genome", referring to its role in conserving stability by preventing genome mutation (Strachan and Read, 1999). The name is due to its molecular mass: it is in the 53 kilodalton fraction of cell proteins.[13] In 1979, scientists discovered a novel protein. This protein, which could bind to a transforming protein (Large T antigen) from Simian Virus 40 (SV40), was more prevalent in cells transformed (immortalized and made potentially tumorigenic) by this virus than in normal cells. The protein and its corresponding gene were named p53, in reference to the mass of the protein (53 kilodalton). The p53 gene is located on chromosome 17 at position p13. Although p53 was the second tumor suppressor to be discovered after Rb, scientists did not understand its true role in the cell until ten years after its discovery. Because p53 was present at increased levels in transformed cells, researchers first believed that it acted as an Oncogene. This belief was supported by initial research. Scientists found that when the p53 gene was transferred into cells, the cells underwent transformation. However, researchers later discovered that the p53 gene that had been transferred was in fact a mutant form of the gene. One normal function of the p53 gene is to prevent cell transformation! Page 4 of 62
    • Cancer p53 jodhpur pharmacy collage 3. DESCRIPTION OF THE GENE 3.1 Genetic Composition Of Cancer p53 The table 1 below shows the genetic composition of p53: UniProt P04637 O70366 RefSeq (mRNA) NM_000546 NM_011640 RefSeq (protein) NP_000537 NP_035770 Location (UCSC) Chr17: 7.51 - 7.53 Mb Chr11: 69.4 - 69.41 Mb The name p53 is in reference to its apparent molecular mass: it runs as a 53 kilodalton (kDa) protein on SDS-PAGE. But based on calculations from its amino acid residues, p53's mass is actually only 43.7kDa. This difference is due to the high number of proline residues in the protein which slow its migration on SDS-PAGE, thus making it appear heavier than it actually is.[5] This effect is observed with p53 from a variety of species, including humans, rodents, frogs, and fish. [16] 3.2 Nomenclature p53 is also known as:  UniProt name: Cellular tumor antigen p53  Antigen NY-CO-13  Transformation-related protein 53 (TRP53)  Tumor suppressor p53 3.3 Cancer p53 in different organisms In humans, p53 is encoded by the TP53 gene located on the short arm of chromosome 17 (17p13.1). TP53 orthologs have been identified in most mammals for which complete genome data are available. Page 5 of 62
    • Cancer p53 jodhpur pharmacy collage In humans, the two most common polymorphisms occur involve the substitution of an Arginine base for a Proline base. This polymorphism arises out of a SNP mutation on the 72 codon, where a guanine bases is replace by a cytosine. [17] For these mammals, the gene is located on different chromosomes:  Chimp and orangutan, chromosome 17  Macaque, chromosome 16  Mouse, chromosome 11  Rat, chromosome 10  Dog, chromosome 5  Cow, chromosome 19  Pig, chromosome 12  Horse, chromosome 11  Opossum, chromosome 3.4 Cancer p53 as an Oncogene p53 protein was first identified in 1979 as a transformation-related protein [21] and a cellular protein which accumulates in the nuclei of cancer cells and binds tightly to the simian virus 40 (SV402) large T antigen [22]. The gene encoding p53 was initially found to have weak oncogenic activity as the p53 protein was observed to be over expressed in mouse and human tumor cells [23]. However, almost 10 years later, researchers discovered that it was a missense mutant of p53 which had originally been considered as wild-type p53 (wt p53) in that previous study, and that the oncogenic properties resulted from a p53 mutation [24,25] ,which was later called ―gain of oncogenic function‖ [26]. By the early 1990s, data from the first p53 knock- out mice provided inarguable evidence in support of the potent tumor suppressor action of wt p53[27]. In subsequent studies, p53 became widely recognized as a tumor suppressor, and the p53 gene became probably the most common site for genetic alterations in human cancers[28,29]. Subsequent research with wt p53 clearly demonstrated that p53 was a biological consequences of p53 Page 6 of 62
    • Cancer p53 jodhpur pharmacy collage activity include cell-cycle regulation, induction of apoptosis, development, differentiation, gene amplification, DNA recombination, chromosomal segregation, and cellular senescence [30]. Presently, p53 is known to play a key role in practically all types of human cancers, and the mutation or loss of the p53 gene can be identified in more than 50% of all human cancer cases worldwide. This significant involvement in oncogenes is extended far beyond the simple role in viral transformation p53 was suspected of playing in earlier investigation.p53 belongs to an unique probe are structurally and functionally related to each other, p53 seems to have evolved in higher organisms to prevent tumor development, whereas p63and p73 have clear roles in normal developmental biology [31- 33]. Because p53 plays a pivotal role in regulation of the cell cycle and induction of apoptosis, there has been enthusiasm about its potential for therapeutic application. It is not surprising that the prominent position p53 plays in tumor development has spurred extensive research into both its basic biologic and clinical aspects. To better understand the relationship between p53 antineoplastic activities and its structure and function, this review focuses on describing biochemical modifications of p53 and p53 mutations. Page 7 of 62
    • Cancer p53 jodhpur pharmacy collage 4. STRUCTURE OF THE GENE 4.1 Description about the structure of cancer p53 Human p53 is 393 amino acids long and has seven domains .These domains with respect to residues are as follows:  N-terminal transcription-activation domain (TAD), also known as activation domain 1 (AD1) which activates transcription factors: residues 1-42.  Activation domain 2 (AD2) important for apoptotic activity: residues 43-63.  Proline Central DNA-binding core domain (DBD) . Contains one zinc atom and several arginine amino acids: residues 100-300.  Nuclear localization signaling domain, residues 316-325.  Homo-oligomerization domain (OD): residues 307-355. Tetramarisation is essential for the activity of p53 in vivo.  C-terminal involved in down regulation of DNA binding of the central domain: residues 356-393.[7]  A tandem of nine-amino-acid transactivation domains (9aaTAD) was identified in the AD1 and AD2 regions of transcription factor p53.[8 ] [9] The p53 protein is a phosphoprotein made of 393 amino acids. It consists of four units (or domains):  A domain that activates transcription factors.  A domain that recognizes specific DNA sequences (core domain).  A domain that is responsible for the tetramarisation of the protein.  A domain that recognized damaged DNA, such as misaligned base pairs or single-stranded DNA. Page 8 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 1 : The structure of the core domain of the p53 protein (light blue) bound to DNA dark blue).The six most frequently Mutated amino acids in human cancer are show in yellow – all are residues important for p53 binding to DNA –Red ball: Zinc atom, {reproduced from cho,Y,et al : (1994) science 1265, 346-355, with kind permission. Page 9 of 62
    • Cancer p53 jodhpur pharmacy collage 4.2 Structure with description about the mutation hot spots in p53 Figure 2 Figure 2: Schematic representation of the p53 structure. p53 contains 393 amino acids, consisting of three functional domains, i.e. an N terminal activation domain, DNA binding domain and C-terminal tetramarisation domain. The N-terminal domain includes transactivation sub domain and a PXXP region that is a Proline-rich fragment. The central DNA binding domain is required for sequence-specific DNA binding and amino acid residues within this domain are frequently mutated in human cancer cells and tumor tissues. TheArg175, Gly245, Arg248, Arg249, Arg273, and Arg282 are reported to be mutation hot spots in various human cancers. The C-terminal region is considered to perform a regulatory function. Residues on this basic C-terminal domain undergo posttranslational modifications including phosphorylation and acetylation. Numbers indicate residue number. NLS, nuclear localization signal sequence; NES, nuclear export signal sequence.[35] Human p53 is a nuclear phosphoprotein of MW 53 kDa, encoded by a 20-kb gene containing 11 exons and 10 introns [16], which is located on the small arm of chromosome 17 [17]. This gene belongs to a highly conserved gene family containing at least two members, p63 and p73. Wild-type p53 protein contains 393 amino acids and is composed of several structural and functional domains : a N-terminus containing an amino-terminal domain (residues 1- Page 10 of 62
    • Cancer p53 jodhpur pharmacy collage 42) and a Proline-rich region with multiple copies of the PXXP sequence (residues 61-94, where X is any amino acid), a central core domain (residues 102-292), and a C terminal region (residues 301-393) containing an oligomerization domain (residues 324-355), a strongly basic carboxyl terminal regulatory domain (residues 363-393), a nuclear localization signal sequence and 3 nuclear export signal sequence [18-20]. The amino- terminal domain is required for transactivation activity and interacts with various transcription factors including acetyltransferases and MDM2 (murinedouble minute 2, which in humans is identified as Hdm2) [21,22]. The Proline-rich region plays a role in p53 stability regulated by MDM2, wherein p53 becomes more susceptible to degradation by MDM2 if this region is deleted [23]. The central core of this protein is made up primarily of the DNA-binding domain required for sequence-specific Unbinding (the consensus sequence contains two copies of the 10-bp motif 5‘-PuPuPuC (A/T)-(T/A) GPyPyPy-3‘, separated by0-13bp) [24]. The basic C-terminus of p53 also functions as a negative regulatory domain [20], and has also been implicated in induction of cell death [25]. According to the allosteric model, in which C-terminal tail of p53 was considered as a negative regulator and may regulate the ability of its core DNA binding domain to lock the DNA binding domain as an latent conformation. Page 11 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 3 – Structural Organization of p53 Core domains occupies four core-nodes of the EM m light orange). Atomic coordinates of the oligomerization domain and two α-helices representing terminus were fitted in to the N/C node of the 3D map. Zn [37] Figure 4: Schematic representations of the corresponding monomer interactions. N/C nodes are represented as blue/magenta joints, the linkers representing N-terminus are in blue, and those for C terminus are in magenta. The core nodes are shown as Page 12 of 62
    • Cancer p53 jodhpur pharmacy collage spheres colored in relation to their corresponding core domains. Grey linkers represent core node to N/C node contacts. [37] Page 13 of 62
    • Cancer p53 jodhpur pharmacy collage 5. MECHANISM OF WORKING FOR CANCER p53 5.1 A brief about working of cancer p53 It plays an important role in cell cycle control and apoptosis. Defective p53 could allow abnormal cells to proliferate, resulting in cancer. As many as 50% of all human tumors contain p53 mutants. In normal cells, the p53 protein level is low. DNA damage and other stress signals may trigger the increase of p53 proteins, which have three major functions: growth arrest, DNA repair and apoptosis (cell death). The growth arrest stops the progression of cell cycle, preventing replication of damaged DNA. During the growth arrest, p53 may activate the transcription of proteins involved in DNA repair. Apoptosis is the "last resort" to avoid proliferation of cells containing abnormal DNA. The cellular concentration of p53 must be tightly regulated. While it can suppress tumors, high level of p53 may accelerate the aging process by excessive apoptosis. The major regulator of p53 is Mdm2, which can trigger the degradation of p53 by the ubiquitin system. [38] 5.2 Target Genes of p53 P53 is a transcriptional activator, regulating the expression of Mdm2 (for its own regulation) and the genes involved in growth arrest, DNA repair and apoptosis. Some important examples are listed below.  Growth arrest: p21, Gadd45, and 14-3-3.  DNA repair: p53R2.  Apoptosis: Bax, Apaf-1, PUMA and NoxA. [39] 5.3 A brief about several mechanisms of cancer p53 In its anti-cancer role, p53 can work through several postulated mechanisms which are as follows:  It can activate DNA repair proteins when DNA has sustained damage. Page 14 of 62
    • Cancer p53 jodhpur pharmacy collage  It can induce growth arrest by holding the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle.)  It can initiate apoptosis, the programmed cell death, if the DNA damage proves to be irreparable. Figure 5: p53 pathway In a normal cell p53 is inactivated by its negative regulator, mdm2. Upon DNA damage or other stress, various pathways will lead to the dissociation of the p53 and mdm2complex.Once activated; p53 will either induce a cell cycle arrest to allow repair and survival of the cell or apoptosis to discard the damage cell. How p53 makes this choice is currently unknown. Activated p53 binds DNA and activates expression of several genes including WAF1/CIP1 encoding for p21. p21 (WAF1) binds to the G1-S/CDK (CDK2) and S/CDK complexes (molecules important for the G1/S transition in the cell cycle) inhibiting their activity. P53 has many anticancer mechanisms, and plays a role in apoptosis, genetic stability, and inhibition of angiogenesis. When p21 (WAF1) is complexed with cdk2 the cell cannot pass through to the next stage of cell division. Mutant p53 can no longer bind DNA in an effective way, and as a Page 15 of 62
    • Cancer p53 jodhpur pharmacy collage consequence the p21 protein is not made available to act as the 'stop signal' for cell division. Thus cells divide uncontrollably, and form tumors. Recent research has also linked the p53 and RB1 pathways, via p14ARF, raising the possibility that the pathways may regulate each other. P53 by regulating LIV has been shown to facilitate implantation in the mouse model and possibly in humans. When p53 expression is stimulated by sunlight, it begins the chain of events leading to tanning. The p53 protein senses DNA damage and can halt progression of the cell cycle in G1 (by blocking the activity of Cdk2). Both copies of the p53 gene must be mutated for this to fail so mutations in p53 are recessive, and p53 qualifies as a tumor suppressor gene. [40] The p53 protein is also a key player in apoptosis, forcing "bad" cells to commit suicide. So, if the cell has only mutant versions of the protein, it can live on — perhaps developing into a cancer. More than half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein. [41] 5.4 activation of p53 by two categories of protein kinases The protein kinases that are known to target this transcriptional activation domain of p53 can be roughly divided into two groups. A first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. A second group of protein kinases (ATR, ATM, CHK1 and CHK2, DNA-PK, CAK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress. Oncogenes also stimulate p53 activation, mediated by the protein p14ARF. 5.5 Mdm 2: its significance in mechanism of action of p53 In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 (also called HDM2 in humans) binds to p53, preventing its action and transports it from the nucleus to the cytosol. Also Mdm2 acts as ubiquitin ligase and Page 16 of 62
    • Cancer p53 jodhpur pharmacy collage covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome. However, ubiquitylation of p53 is reversible. A ubiquitin specific protease, USP7 (or HAUSP), can cleave ubiquitin off p53, thereby protecting it from proteasome-dependent degradation. This is one means by which p53 is stabilized in response to oncogenic insults. Phosphorylation of the N-terminal end of p53 by the above-mentioned protein kinases disrupts Mdm2-binding. Other proteins, such as Pin1, are then recruited to p53 and induce a conformational change in p53 which prevents Mdm2- binding even more. Phosphorylation also allows for binding of transcriptional co activators, like p300 or PCAF, which then acetylates the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes. Deacetylate enzymes, such as Sirt1 and Sirt7, can deacetylate p53, leading to an inhibition of apoptosis. [19] Some oncogenes can also stimulate the transcription of proteins which bind to MDM2 and inhibit its activity. As mentioned above, p53 is mainly regulated by Mdm2. The regulation mechanism is illustrated in the following figure 6. Page 17 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 6.0 Regulation of p53 From figure 6 we come to know about the following points:  Expression of Mdm2 is activated by p53.  Binding of p53 by Mdm2 can trigger the degradation of p53 via the ubiquitin system.  Phosphorylation of p53 at Ser15, Thr18 or Ser20 will disrupt its binding with Mdm2. In normal cells, these three residues are not phosphorylated, and p53 is maintained at low level by Mdm2. Page 18 of 62
    • Cancer p53 jodhpur pharmacy collage  DNA damage may activate protein kinase (such as ATM, DNA- PK, or CHK2) to phosphorylate p53 at one of these three residues, thereby increasing p53 level. Since Mdm2 expression is activated by p53, the increase of p53 also increases Mdm2, but they have no effect while p53 is phosphorylated. After the DNA damage is repaired, the ATM kinase is no longer active. p53 will be quickly dephosphorylated and destroyed by the accumulated Mdm2. [42] 5.6 The regulation of p53’s level in cell Under normal circumstances, p53 is maintained at very low concentrations within the cells and exists mainly in an inactive latent form [43]. During the cell cycle progression, the low basal level of wt p53 has to be precisely controlled [44]. In normally growing cells, the half-life of p53 is limited to Minutes, whereas cellular stress or exposure to DNA damaging agents prolongs it to hours [45] . Increased levels of the p53 protein are primarily regulated through lengthening of its half-life. The level of p53 and its activities in the cell depend on the cell‘s situation and extracellular stimuli. The figure 7 shows the p53 pathway and associated genes in apoptosis. Page 19 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 7: p53-associated genes and pathways involved in apoptotic cell death. p53 induces apoptosis mainly via two pathways: extrinsic and intrinsic pathways. The p53- associated extrinsic pathway is mainly executed by activating caspase 8 to induce apoptosis, whereas the p53-associated intrinsic pathway is almost executed by influencing mitochondrial proteins, by which activate caspase 9 to induce apoptosis. In addition p53 may directly activate Apaf-1 to induce apoptosis 5.7 Genes involved in regulating p53 level in cell and its activity The regulation of p53 level and activity involves a complex network of a multitude of cellular proteins as shown in figure 7 , including HPV16E6[46] , WT-1 [94], E1B/E4[47] , SV40 ,T-antigen[49,50] ,MDM2[51,52] , JNK[53] , Pirh2[54,55], and PARP-1[62,57]. Moreover, p53 can switch between a latent and an active form in its function as a transcription factor. The binding of SV40 T antigen, WT1 or E1B/E4 with p53 increases its stability, whereas the association of E6 or MDM2 with p53 accelerates its degradation. MDM2 is an important related protein, which is the product of a p53 inducible gene. The Page 20 of 62
    • Cancer p53 jodhpur pharmacy collage importance of MDM2 in the regulation of p53 levels is demonstrated by the fact that disruption of the MDM2 gene is lethal in early embryos, whereas the concurrent inactivation of the p53 gene rescues the animal from a lethal consequence [58]. MDM2 inhibits p53 activity by blocking its transcriptional activity, favoring its nuclear export and stimulating its degradation. MDM2 protein has been found to play an additional role in blocking the interaction of p53 with the transcriptional apparatus by binding to and shielding the transactivation domain ofp53 within its N-terminus[52,59] .MDM2 protein possesses intrinsic E3 ubiquitin ligase activity and mediates both the ubiquitylation and proteasome dependent degradation of p53[60] . Ubiquitinated p53 is exported to the cytoplasm, thereby moving it away from its site of action and promoting its rapid degradation by the proteosome [51]. MDM2 can also recruit the histones deacetylase1 (HDAC1) to deacetylate key lysine residues in the C Terminus of p53, thus making them available for ubiquitination. The MDM2 gene itself also contains ap53-dependentpromoter and is transcriptionally regulated by p53 following challenge of the cell by various stresses [61]. In this fashion, the p53protein regulates the MDM2 gene at the level transcription and the MDM2 protein regulates the p53 protein at the level of its activity, and an autoregulatory feedback loop is established that regulates both the activity of the p53 protein and the expression of the MDM2 gene. In this sequence of events, it is wtp53 that is targeted by MDM2 for degradation, whereas p53 is outside of this negative feedback loop and accumulates to high levels in cancer cells. It is becoming evident that a number of mechanisms exist to abolish MDM2-mediated degradation of p53, thereby allowing the maintenance of a p53 response initiated by various genotoxic stimuli [20, 30]. Under stress conditions, distinct signaling pathways can be activated to prevent p53 from ubiquitylation and degradation through post translational modifications and abolishment of MDM2 activity. Several regulators of p53 have been identified recently, such as the positive regulator PML (promyelocytic leukemia protein) [66, 67] and the negative regulators YY1 (Yin Yang1) [68], survivin [64] and PLD (phospholipase D)[65]. All these regulators appear to influence p53 through MDM2, and they all appear to affect the transcriptional activity of p53. Page 21 of 62
    • Cancer p53 jodhpur pharmacy collage 6. ROLE OF P53 IN CANCER TREATMENT 6.1 Role of P53 and MDM2 in Treatment Response of Human Germ Cell Tumors PURPOSE: Testicular germ cell tumors (TGCTs) of adolescents and adults are very sensitive to systemic treatment. The exquisite chemo sensitivity of these cancers has been attributed to a high level of wild-type P53. RESULTS: Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%, and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the positive TGCTs, most tumors, i.e., 49%, 41%, and 33%, showed 1% to 10% positive nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis revealed only one silent P53 mutation in one of the responding patients. All embryonal carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA, while a heterogeneous pattern was found for the other histological components. Amplification of MDM2 was detected in one out of 12 embryonal carcinomas. CONCLUSION: The results of this abstract show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and only inactivation of P53 is not a very exact cause in the development of cisplatin resistance. 6.2 Adenovirus-based p53 gene therapy in ovarian cancer Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, an adenovirus vector containing the wild-type p53 gene was constructed. The ability of this adenovirus construct (Ad-CMV- p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus Page 22 of 62
    • Cancer p53 jodhpur pharmacy collage containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad- CMV-p53 showed expression of wild-type p53 protein. Conclusion When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells shows beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer. [75] 6.3 Functional inactivation and structural mutation in p53 causes liver cancer Structural mutations in the p53 gene are seen in virtually every form of human cancer. To determine whether such mutations are important for initiating tumorigenesis, the hepatocellular carcinoma have been studied, in which most cases are associated with chronic hepatitis B virus infections. Using a transgenic mouse model where expression of a single HBV gene product, the HBx protein, induces progressive changes in the liver, showed that tumour development correlates precisely with p53 binding to HBx in the cytoplasm and complete blockage of p53 entry into the nucleus. Conclusion Page 23 of 62
    • Cancer p53 jodhpur pharmacy collage Analysis of tumour cell DNA shows no evidence for p53 mutation, except in advanced tumors where a small proportion of cells may have acquired specific base substitutions. The results suggest that genetic changes in p53 are late events which may contribute to tumour progression [76-82]. 6.4 Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53 Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37°C and a wild-type like p53 at 32°C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfectlon of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfectlon experiments utilizing the neomycin resistance gene only (SKN). Conclusion Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that are associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32°C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32°C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32°C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not In SKN cells. In both clones Gas1 mRNA was not detected either at 37°C or 32°C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37°C) or wild type p53 (SK23a and SK9 kept at 32°C) on the mechanism of response of cancer cells to DNA damaging agents. [83] Page 24 of 62
    • Cancer p53 jodhpur pharmacy collage 6.5 p53 alterations in recurrent squamous cell cancer of the head and neck refractory to radiotherapy Abstract The aim of the study was to determine the incidence of p53 alterations by mutation, deletion or inactivation by mdm2 or human papillomavirus (HPV) infection in recurrent squamous cell cancer of the head and neck (SCCHN) refractory to radiotherapy. Twenty- two tumours were studied. The p53 status of each tumour was analysed by sequencing of exons 4–9 and by immunohistochemistry. Mdm2 expression was assessed by immunohistochemistry and HPV infection was assessed by polymerase chain reaction of tumour DNA for HPV 16, 18 and 33. Fifteen (68%) of the 22 tumors studied had p53 mutations, while seven had wild-type p53 sequence. p53 immunohistochemistry correlated with the type of mutation. HPV DNA was detected in 8 (36%) tumors and all were of serotype HPV 16. Of these, five were in tumors with mutant p53 and three were in tumors with wild-type p53. Conclusion Mdm2 over expression was detected in 11 (50%) tumors. Of these, seven were in tumors with mutant p53 and four were in tumors with wild-type p53. Overall, 21 of the 22 tumors had p53 alterations either by mutation, deletion or inactivation by mdm2 or HPV. In this study, the overall incidence of p53 inactivation in recurrent head and neck cancer was very high at 95%. The main mechanism of inactivation was gene mutation or deletion which occurred in 15 of the 22 tumors studied. In addition, six of the seven tumors with wild-type p53 sequence had either HPV 16 DNA, over expression of mdm2 or both which suggested that these tumors had p53 inactivation by these mechanisms. This high incidence of p53 dysfunction is one factor which could account for the poor response of these tumors to radiotherapy and chemotherapy. Therefore, new therapies for recurrent SCCHN which either act in a p53 independent pathway, or which restore p53 function may be beneficial in this disease. [84] 6.6 Endocrine-Related Cancer Page 25 of 62
    • Cancer p53 jodhpur pharmacy collage Background The scientists Lane and Harris have reviewed that the tumour suppressor gene p53 and its protein control critical cellular functions are involved in apoptosis and in the control of the cell cycle. The encoded protein consists of 393 amino acids giving a molecular mass of 53 000 Daltons. Kirsch and Kastan reviewed that the N terminal part of the protein is involved in transcription control, the middle portion is responsible for the DNA binding and the carboxy-terminal third of the protein facilitates the tetramarisation of the protein, which is claimed to be required for its function. Garber et al and Serrano et al in 1997 found that the p53 gene can be activated via the ataxia telangectasia gene (ATM) by carcinogens, cytostatics, radiation, ultraviolet light, and hypoxia or by an Oncogene. When cells acquire irreparable damage, the reparable damage the cell cycle is retarded via p53- initiated downstream activationof the cyclin-dependent kinases. This results in inhibition of the cyclins, together with interaction with the retinoblastoma gene product aiming at controlling the cell cycle at specific checkpoints.The normal p53 function can be inactivated by somatic and germ line mutations, binding to the Oncogene murinedouble minute (MDM2) and binding to different viraloncoproteins (humanpapilloma virus protein E6, SV40large T antigen, hepatitis B viral X protein, adenovirus protein E1A) which was reviewed in Harris 1996. Clarke et al. 1993, Lowe et al.1993, 1994, O‘Connor et al. 1993, Fan et al. 1994, Lim etal. 1994demonstrated that somatic mutation of the p53 gene is, so far, the most common geneticabnormality described in humancancer.In pre-clinical model systems it was demonstrated thattumours with wild-type p53 status responded better tocertain ontological therapeutic modalitiesthan tumourswith an altered p53 status . Clarke et al.1993 say that despite these straight forward findings it is also obvious that radiation and certain cytostaticsmay alsoinduce apoptosis via a p53-independent pathway which was reviewed in Beck & Dalton 1997. However, the issue is complex, sincemutant p53 has been claimedto interfere ‗with the p53-independent pathways of apoptosis‘ (Li et al. 1998). Jost et al. 1997, Kaghad et al.1997, Osada et al.1998, Trink et al demonstrate that p40, p51 and p73 have more or less sequence homology with p53 .These genes are now grouped togetherin the p53family. How they are activated and may replace andfunction whenp53 is mutated is not known. These aspectsmay be important both for p53dependent and-independent apoptosis. [85] Page 26 of 62
    • Cancer p53 jodhpur pharmacy collage 6.7 Clinical studies of p53 in treatment and benefit of breast cancer patients Abstract This review article focuses on discussing p53 inrelation to its predictive potential. So far, no firm conclusions can be made based on the articlesstudied. This may be, in part, because many studies have usedless than optimal techniques fordetermination of the p53 status, together with the fact that the studies lacked power to detect an Endocrine- Related Cancer (1999) 6 51-59potential difference in outcome from specific therapy in relation to p53 status.Bergh: p53 treatment in breast cancer patients aspect of p53. In comparative studies, the use ofimmunohistochemistry and another protein measurement will result in false negative and false positive results. Conclusion P53 has critical functions for the control of the cell cycle and apoptosis. Cytostatics, tamoxifen and radiation may induce apoptosis via p53-dependent and –independent pathways. The clinical data with reference to the potential predictive value of p53 are still conflicting, which is due partly to suboptimal methods for determination of the p53status, and partly to too small studies combined withselected patient materials. The data so far indicate that CMF-based regimens and tamoxifen may be suboptimalfor patients with mutant p53 andpostoperativeradiotherapy may be extra beneficial for breast cancerswith mutant p53. For the future, larger studies onpopulation-based cohorts using optimalmethods for p53measurements are warranted, and ideally the studies should have patient populations. [86] 6.8 Boehringer Ingelheim and Priaxon announce a collaboration to research and develop novel treatments for cancer Page 27 of 62
    • Cancer p53 jodhpur pharmacy collage The two Companies to advance mdm2/p53 inhibitors, which hold potential to address various tumor types: Munich, Ingelheim/Germany, 18 January 2010 - Boehringer Ingelheim and Priaxon entered into a worldwide collaboration to research and develop mdm2/p53 inhibitors for the treatment of cancer. Priaxon is providing its innovative and proprietary small molecule drug discovery expertise which is particularly suited to investigate inhibition of protein-protein interactions. P53 is a human tumor suppressor protein. It has been shown that in tumors with wild-type p53, the restoration of p53 tumor-suppressive functions can be achieved by blocking a cellular interaction of mdm2 1 and p53. This may reactivate the ―genome guardian‖ function of p53 and is therefore an interesting approach for treating under the terms of the collaboration and license agreement, Boehringer Ingelheim will lead development and commercialisation of the potential mdm2/p53 inhibitor products to capitalise on its global marketing and sales expertise. Boehringer Ingelheim will pay significant up-front and near-term payments to Priaxon including research funding to support further discovery efforts. In addition, Priaxon will be eligible to receive from Boehringer Ingelheim EUR 86 million in milestone payments upon achievement of certain development, regulatory and commercial milestones as well as royalties on potential future net sales of products. About mdm2/p53 Inhibition The human p53 tumor suppressor protein has been one of the most investigated proteins in cancer research due to the fact that loss of p53 function through mutation and/or deregulation is involved in about 50% of all human cancers. The role of p53 in controlling the cell cycle and monitoring the integrity of the genome has made it known as the ―guardian of the genome‖. Besides the functional loss of p53 through mutation, it can also be inactivated by the over expression or amplification of MDM2 (murine double minute 2), which is the case in many p53 wild-type tumors. Thus, disruption of the MDM2–p53 interaction is considered a novel therapeutic strategy for cancer cells that still are endowed with wild-type p53, and a variety of small molecule drug like compounds haveto the p53 binding site of MDM2. About Priaxon Page 28 of 62
    • Cancer p53 jodhpur pharmacy collage Priaxon is an emerging pharmaceutical company building a pipeline of novel drug candidates in different therapeutic fields, but mainly focusing on protein-protein interactions in oncology and other diseases. The goal is to discover and develop candidates for validated but hard-to-drug targets using two orthogonal drug discovery platforms. About Boehringer Ingelheim The Boehringer Ingelheim group is one of the world‘s 20 leading pharmaceutical companies. Headquartered in Ingelheim, Germany, it operates globally with 138 affiliates in 47 countries and 41,300 employees. Since it was founded in 1885, the independent, family-owned company has been committed to researching, developing, manufacturing and marketing novel products of high therapeveterinary medicine. In 2008, Boehringer Ingelheim posted net sales of 11.6 billion euro while spending one fifth of net sales in its largest business segment Prescription Medicines on research and development. Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. [87] 6.10 Regulation of cancer stem cells by p53 The hypothesis those cancer stem cells are responsible for the chemoresistant and metastatic phenotypes of many breast cancers has gained support using cell sorting strategies to enrich the tumor-initiating population of cells. The mechanisms regulating the cancer stem cell pool, however, are less clear. Two recent publications suggest that loss of p53 permits expansion of presumptive cancer stem cells in mouse mammary tumors and in human breast celllines.These results add restriction of cancer stem cells as a new tumor suppressor activity attributed to p53. The recent identification and characterization of stem cells in a variety of adult tissues has led interest in there of stem cells in cancers.Cancer stem cells are hypothesized to be Page 29 of 62
    • Cancer p53 jodhpur pharmacy collage a small population of cells within a tumor that are capable of self-renewal and that can undergo differentiation to generate the phenotypic heterogeneity observed in tumors.Contemporary methods to study cancer stem cells have often used cell surface markers to enrich the subset of cells capable of initiating a tumor upon transplantation into an appropriate host. Molecular pays that limit expansion of the tumor-initiating cell population could be targeted to eradicate tumors.Using mammary tumors arising spontaneously from transplants of BALB/c-Trp53-/- mammary epithelium, Zhang and coworkers show that cells expressing markers of mouse mammary stem cells (lin- /CD29hi/CD24hi) had a greater tumor-initiating frequency. This observation was consistent among tumors with heterogeneous expression of markers for the luminal epithelium and the basal epithelium. The lin-/CD29hi/CD24hi population shared additional features of mammary stem cells, including radiation resistance and the formation of secondary mammospheres. But how might loss of p53 lead to formation or expansion of the tumor-initiating pool? Using a unique culture model of luminal breast epithelial cells (BPEC-T), Godar and coworkers demonstrate that p53 binds to the promoter of CD44, a commonly used marker of cancer stem cells, and represses CD44 expression. Constitutive expression of CD44 blocked p53-dependent apoptosis and rendered cells resistant to doxorubicin. Conversely, suppression of CD44 expression restricted tumor-initiating cells. Conclusion These results link the loss of p53 function to increased expression of CD44, which promotes expansion of tumor-initiating cells purified in tumors. The p53 protein appears to play a similar role in embryonic stem cells, where p53 represses expression of Nanog – which limits the pool of pluripotent cells. In contrast, loss of p53 extends the repopulating activity of tissue-specific stem cells. Disruption of BRCA1 also allows expansion of breast stem cells. The restriction of stem cells may therefore be a fundamental pathway for tumor suppression. While expansion of the tumor-initiating cell population in p53-deficient mammary epithelial cells is consistent in both mouse mammary and human breast epithelial cells, the role of CD44 is not. Although loss of p53 expression resulted in increased levels of CD44 protein in BPEC-T cells and in basal mammary epithelium of Trp53-/- mice , there was no enrichment for tumor-initiating cells within the CD44+/CD24-population in Page 30 of 62
    • Cancer p53 jodhpur pharmacy collage BALB/c-Trp53-/- mammary tumors . This apparent discrepancy points to heterogeneity in the expression of markers among cancer stem cells. In mammary tumors from Brca1ΔExon11/Trp53+/- mice, two discrete tumor-initiating populations were identified that express CD44+/CD24-or CD133+. As coexpression of CD44 and CD133 was not detected in these pools of cells, it appears that CD44 is not essential for sustaining the pool of cancer stem cells. Indeed, p53 represses expression of more than 20 target genes that may contribute to maintenance of the pool of tumor-initiating cells. Genes such as Nanog may have direct actions in supporting self-renewal of cancer stem cells, allowing the pool to expand. Loss of p53 would also allow increased expression of the multidrug-resistance gene (ABCB1 or MDR1) that renders cells resistant to chemotherapies. Similarly, both increased proliferation and decreased apoptosis would be expected to result from de-repression of CDC25C and BIRC5/Survivin when p53 function is disrupted. CD44 may therefore be only one mechanism by which p53 may act to restrict the tumor-initiating population of cancer cells. It is clear that that p53 plays a pivotal role in tumor suppression. Mutation and loss of function of p53 are among the most common alterations in epithelial cancers., and gene expression signatures associated with dysfunctional p53 have been shown to predict patient survival .The p53 protein regulates a variety of pathways (cell cycle arrest, apoptosis, DNA repair, senescence and autophagy) that can contribute to suppression of tumors. The publications by Zhang and colleagues and by Godar and colleagues now add suppression of cancer stem cells as an additional activity by which p53 can inhibit tumors. On the one hand, loss of p53 may promote genetic instability – resulting in plasticity of phenotypes due to random mutations and clonal evolution. . Page 31 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 8: Loss of p53 function and effects on tumor heterogeneity. In normal epithelia, p53 represses expression of potential oncogenes (for example, CD44, NANOG, BIRC5, CDC25C) as well as transcriptionally activating tumor suppressor pathways [10]. Loss of p53 (more ...) In this model, the behavior of the p53-deficient cancer cells would be stochastic and would require therapeutics targeting multiple oncogenic pathways. If the apparent phenotypic plasticity of p53-deficient breast tumors is due to the expansion of the cancer stem cell pool, however, therapies targeting the self-renewal pathways may be extremely effective. Loss of p53 function in breast tumors is strongly correlated with the basal-like gene expression signatures. This suggests that either these tumors originate from breast stem cells or that loss of p53 allows cancer cells to acquire characteristics of stem cells. These results favor the possibility that p53 deficiency allows expansion of cancer stem cells and that the expression profiles of tumor-initiating cells will identify effective therapeutic targets. [88] 6.9 Role in disease Page 32 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 9: . If the TP53 gene is damaged, tumor suppression is severely reduced. People who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disease known as Li-Fraumeni syndrome. The TP53 gene can also be damaged in cells by mutagens (chemicals, radiation, or viruses), increasing the likelihood that the cell will begin decontrolled division. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Increasing the amount of p53, which may initially seem a good way to treat tumors or prevent them from spreading, is in actuality not a usable method of treatment, since it can cause premature aging. However, restoring endogenous p53 function holds a lot of promise. Loss of p53 creates genomic instability that most often results in the aneuploidy phenotype. Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such example, the Human papillomavirus (HPV), encodes a protein, E6, which binds the p53 protein and inactivates it. This, in synergy with the inactivation of another cell cycle regulator, p105RB, allows for repeated cell division manifested in the clinical disease of warts. Infection by oncogenic HPV types, especially HPV16, can also lead to progression from a benign wart to low or high-grade cervical dysplasia which is reversible forms of precancerous lesions. Persistent infection over the years causes irreversible changes leading to Carcinoma in situ and eventually invasive cervical cancer. This results from the Page 33 of 62
    • Cancer p53 jodhpur pharmacy collage effects of HPV genes, particularly those encoding E6 and E7, which are the two viral oncoproteins that are preferentially retained and expressed in cervical cancers by integration of the viral DNA into the host genome. In healthy humans, the p53 protein is continually produced and degraded in the cell. The degradation of the p53 protein is, as mentioned, associated with MDM2 binding. In a negative feedback loop MDM2 is itself induced by the p53 protein. However mutant p53 proteins often don't induce MDM2, and are thus able to accumulate at very high concentrations. Worse, mutant p53 protein itself can inhibit normal p53 protein levels.[89] 6.10 P53 Gene Therapy: A Potential Panacea To Cancer The extensive work focuses on gene therapy as a novel method to cure cancer [ovarian &breast], especially p53 gene having tremendous potential used as a suppressor gene to kill the tumor cells, their types, roles and use of p53. It focuses on the mechanism of p53 apoptosis [programmed cell death]. It suggests advanced therapies in combination of chemotherapy with gene therapy. It also shed light on various current research viral & non viral vectors used in p53 gene therapy. The figure 11 shows role of p53 in apoptosis. Page 34 of 62
    • Cancer p53 jodhpur pharmacy collage Figure 10: Role of apoptosis In each of these diverse areas implicates immense potential manipulation of apoptosis to treat disease. Research is already underway to harness apoptosis as a therapeutic tool in modern medicine. As chemotherapy show disadvantage of regrowth of cell, metastasis after surgery treatment, chemoresistance to tumor, gene therapy has been a revolutionary step towards cancer treatment. Genetic correction strategies are presently being developed and tested in animal models for human malignancies and in early patient trials. The cancer susceptibility genes p53 have been tested in ovarian & breast cancer patients, , and have shown some potential for this antitumor strategy. P53 gene therapy may be Page 35 of 62
    • Cancer p53 jodhpur pharmacy collage effective even against tumors that lack p53 mutations, because p53 may function as a growth inhibitor in a variety of gene transfer settings. Key problems at present include the degradation of vector by the immune system and a need for higher levels of gene transduction. Solutions will require the development of improved vectors, improved vector delivery systems, and the fine-tuning of human gene therapy in appropriate models of human cancer. [90] 6.11 Role of p53 Gene in Metabolism Regulation in Patients with Li-Fraumeni Syndrome Purpose This study will examine metabolic and biological factors in people with Li-Fraumeni syndrome, a rare hereditary disorder that greatly increases a person's susceptibility to cancer. Patients have a mutation in the p53 tumor suppressor gene, which normally helps control cell growth. This gene may control metabolism as well as cancer susceptibility, and the study findings may help improve our understanding of not only cancer but also other conditions, such as cardiovascular function.[91] 6.12 The role of p53 in treatment responses of lung cancer Abstract Resistance to radio- and chemotherapy is a major problem in treatment responses of lung cancer. In this disease, biological markers, that can be predictive of response to treatment for guiding clinical practice, still need to be validated. Radiotherapy and most Page 36 of 62
    • Cancer p53 jodhpur pharmacy collage chemotherapeutic agents directly target DNA and in response to such therapies, p53 functions as a coordinator of the DNA repair process, cell cycle arrest, and apoptosis. In fact, it participates in the main DNA repair systems operative in cells, including NHEJ, HRR, NER, BER, and MMR. Given the high p53 mutation frequency in lung cancer which likely impairs some of the p53-mediated functions, a role of p53 as a predictive marker for treatment responses has been suggested. In this review, we summarize the conflicting results coming from preclinical and clinical studies on the role of p53 as a predictive marker of responses to chemotherapy or radiotherapy in lung cancer. [92] 6.13 The Association of p53 with Specific Cancers Germline (inherited) mutations of the p53 gene are associated with the rare inherited cancers classified under the Li-Fraumeni syndrome (LFS). Somatic p53 genetic mutations have been shown to be involved in tumors of the anus, bone, bladder, brain, breast, colon, cervix, esophagus, stomach, liver, lung, lymphoid system, ovary, prostate and skin. A brief look at the p53 gene's association with certain cancers is presented below. Lung cancer Benzo[a]pyrene is a mutagen found in cigarette smoke. This chemical binds to DNA and ultimately can cause G (guanine) to T (thymine) substitutions in DNA. Other chemicals in cigarette smoke have been shown to produce C (cytosine) to A (adenine) changes. When these occur in the p53 gene, the mutations can cripple the p53 protein, disrupting its tumor-suppressing function. Liver cancer Page 37 of 62
    • Cancer p53 jodhpur pharmacy collage Two major causes of liver cancer are infection with the Hepatitis-B virus and exposure to aflatoxin, a mutagen produced by a mold that grows on improperly stored grains and food crops, specifically wet corn. Aflatoxin, like benzopyrene, may change the gene that codes for p53, thereby disrupting the tumor-suppressing ability of p53.The Hepatitis-B virus works to inactivate p53 in a different way; it produces a protein that has the ability to bind p53 and prevent it from interacting effectively with its target genes. Skin cancer The ultraviolet (UV) rays in sunlight can cause damage to DNA. If the DNA in a skin cell is damaged beyond repair, the p53 protein can induce cell death. However, if the UV light causes a mutation in the p53 gene rendering the protein nonfunctional, the damaged cell may reproduce and potentially lead to the formation of a cancerous growth. Cervical cancer Human papillomavirus (HPV) is a sexually transmitted virus that can infect cervical cells. Once inside the cell, the virus produces a protein that binds to p53 and causes the p53 protein to be degraded. The result of this degradation is a decrease in available p53 protein and a loss of functinal p53 activity. Breast cancer In many breast cancers the p53 gene appears to be normal. However, it has been shown that in some cases the protein MDM2 is enhanced in the cells and binds to the p53 protein, inhibiting its antitumor activity. This allows for the growth of malignant breast cells and inhibits the p53 induced apoptotic pathway.[93] Page 38 of 62
    • Cancer p53 jodhpur pharmacy collage 7. POTENTIAL THERAPEUTIC USE AND SIGNIFICANCE OF CANCER p53 7.1 Trifluorothymidine Induces Cell Death Independently of p53 Abstract Trifluorothymidine (TFT), a potent anticancer agent, inhibits thymidylate synthase (TS) and is incorporated into the DNA, both events resulting in cell death. Cell death induction related to DNA damage often involves activation of p53. The role of p53 was determined in TFT cytotoxicity and cell death induction, using, respectively, the sulforhodamine B- assay and FACS analysis, in a panel of cell lines with either wild type, inactive, or mutated p53. Neither TFT cytotoxicity nor cell death induction changed with TFT exposure in cell lines with wt, inactive or mutated p53. Conclusion: sensitivity to TFT is not dependent on the expression of wt p53. In-vitro introduction of p53 in to p53-deficient cells has been shown to cause rapid death of cancer cells or prevention of further division. The rationale for developing therapeutics targeting p53 is that "the most effective way of destroying a network is to attack its most connected nodes". P53 is extremely well connected (in network terminology it is a hub) and knocking it out cripples the normal functioning of the cell. This can be seen as 50% of cancers have missense point mutations in the p53 gene, these mutations impair its anti- cancer gene inducing effects. Restoring its function would be a major step in curing many cancers (Vogelstein et al 2000). Various strategies have been proposed to restore p53 function in cancer cells like the one given by Blagosklonny in 2002. A number of groups have found molecules which appear to restore proper tumour suppressor activity of p53 in vitro. These work by altering the conformation of mutant conformation of p53 back to an active form. So far, no molecules have shown to induce biological responses, but some may be lead compounds for more biologically active agents. A promising target for anti-cancer drugs is the molecular chaperone Hsp90, which interacts with p53 in vivo. Page 39 of 62
    • Cancer p53 jodhpur pharmacy collage Adenoviruses rely on their host cells to replicate; they do this by secreting proteins which compel the host to replicate the viral DNA. Adenoviruses have been implicated in cancer- causing diseases, but in a twist it is now modified viruses which are being used in cancer therapy. ONYX-015 (dl1520, CI-1042) is a modified adenovirus which selectively replicates in p53-deficient cancer cells but not normal cells says Bischoff in 1996. It is modified from a virus that expresses the early region protein, E1B, which binds to and inactivates p53. P53 suppression is necessary for the virus to replicate. In the modified version of the virus E1B has been deleted. It was hoped that the viruses would select tumour cells, replicate and spread to other surrounding malignant tissue thus increasing distribution and efficacy. The cells which the adenovirus replicates in are lysed and so the tumour dies. Preclinical trials using the ONYX-015 virus on mice were promising however clinical trials have been less so. No objective responses have been seen except when the virus was used in combination with chemotherapy says McCormick in 2001. This may be due to the discovery that E1B has been found to have other functions vital to the virus. Additionally its specificity has been undermined by findings showing that the virus is able replicate in some cells with wild-type p53. The failure of the virus to produce clinical benefits may in large part be due to extensive fibrotic tissue hindering virus distribution around the tumour says McCormick in 2001. [95] 7.2 Therapeutic applications of p53 The insights which have been provided by p53 researchover the years have been for improvement of diagnostic techniques, accuracy of prognosis, and treatment of cancer.Since about half of all human tumors have an abnormal p53 which can occur early in carcinogenesis, and since post translational modifications of p53 can reflect the type and magnitude of cellular stress [114], p53 can be a useful biomarkerin carcinogenesis. Indeed, p53 has been used as a molecular signature to study both target tissues and surrogate fluids such as blood in high-risk cancer populations [173]. Mutated p53 protein accumulation and posttranslational modification endpoints could also prove useful Page 40 of 62
    • Cancer p53 jodhpur pharmacy collage instudying the efficacy of chemopreventive agents [38].As p53 plays a key role in the cellular response to stress; it serves as a major barrier to tumorigenesis. This obstacle has to be removed in order for tumor development to proceedand restoration of wt p53 function is thus a potential key in anticancer therapies. Since MDM2 is an importantnegative regulator of p53, MDM2 hyperactivity may inhibitthe function of p53 and lead to the development of a widevariety of cancers. For example, 30% of human sarcomasshow no p53 mutations, but have an overexpressed MDM2gene [174]. It is believed that inhibiting the E3 activity ofMDM2 and blocking the interaction of p53 with MDM2 are potential effective strategies for killing certain tumor cellsselectively by restoring the function of wt p53 [41]. Therefore, many studies have focused on the p53- MDM2 interactionas the basis of a drug development strategy. A series ofsmall molecule inhibitors have been developed, and some ofthese can bind to MDM2 and block its interaction with p53, including peptides that have been shown to elevate the levelsof p53 protein and its transcriptional activity and triggerp53-dependent apoptosis in tumor cells [175,176]. Class ofsmall molecules named nutlins have been identified to block p53/MDM2 interaction in vitro and in vivo. Treatment of tumor cells with nutlins results in induction of p53 and its target genes and triggering of apoptosis. Recently, a novelseries of benzodiazepinedione antagonists of the p53/MDM2interaction have been discovered which increase the transcriptionof p53 target genes and decrease proliferation oftumor cells expressing wt p53 [177]. One study suggests that antisense oligodeoxynucleotides targeted against MDM2and p21Waf1/Cip1 could be employed in a potential therapeutic strategy sensitizing tumor cells to certain antineoplastic agents [178]. One of the major concerns about blocking thep53/MDM2 interaction for use in treatment of cancer was the idea that activation of p53 might be toxic to normal tissues.However, certain data suggest that the mechanisms governing p53 activity in tumor cells and normal cells are quite different, so the different effects of p53 in reactivating different molecules in tumor cells and normal cells might providea molecular basis for a therapy without the need for tumor targeting [179].Another factor in p53 inactivation is the presence of the human papilloma virus (HPV). In cervical carcinomas, p53 istargeted by HPV encoded E6 protein, which potentiates p53degradation and inactivates its function in 90% of cervical cancers [180]. Drugs that inhibit E6 should promote p53reactivation and thus have selective therapeutic effect. ItE6 expression, stabilize p53 and induce apoptosis in a model system of cultured cells [181]. Interestingly, both drugs can inhibit MDM2-mediated inactivation of p53, Page 41 of 62
    • Cancer p53 jodhpur pharmacy collage possiblyvia inhibition of p53 ubiquitylation (leptomycin B) or by Decreasing MDM2 gene transcription (actinomycin D) [182]. Because the apoptotic function of p53 is critical for tumorsuppression, induction of apoptotic pathways through p53-induced apoptotic targets may be an attractive strategy for anti-cancer treatment. Furthermore, the p53 apoptotic targets,unlike p53, are rarely mutated in human cancers [30].Some of the p53 apoptotic targets, such as bax, Puma,p53AIP1, Noxa and others could potentially be used as targets transfer of bax can act synergistically with chemotherapy to induce apoptosis in tumors[184]. A recent study has demonstrated that siRNA targeting of survivin, a negative regulator of apoptosis which is downregulated by p53, could be potentially useful for increasing sensitivity to anticancer drugs, especially in drug-resistant cells with mutated p53 [185]. However, the effects of p53-dependent apoptosis are not always favorable for clinical use, and so the inhibitors of p53-mediated apoptosis might be used to Significance of PML and p53 protein as molecular prognostic markers of gallbladder carcinomas detection and prognostic prediction, but may also serve as potential therapeutic targets.Inorder to identify reliable molecular markers for prognostic prediction in gall bladder carcinoma (GBC), we evaluated the immunohistochemical expression of 15proteins, namely p53, p27, p16, RB, Smad4, PTEN, FHIT, GSTP1, MGMT, E-cadherin,nm23, CD44, TIMP3, S100A4, and promyelocytic leukemia (PML) in 138 cases of protein. Over expression of p53 and S100A4, and loss of p27, p16, RB, Smad4, FHIT, Ecadherin and PML expression were associated with poor survival. In particular, PML andp53 showed considerable potential as independent prognostic markers. Patients with normal PMLand p53 expression displayed favorable outcomes, compared to those showingabnormal expression of either or both proteins (49%vs. 23% in a 5-year survival rate; 60 monthsvs. 11 months in median survival, respectively;P=0.009). Thus, PML and p53 are potential candidates for development as clinically applicable molecular prognostic markers of GBC, and may be effective therapeutic targets for the disease in the future. [96] 7.3 Clinical significance of p53 alterations in surgically treated prostate cancers Abstract Page 42 of 62
    • Cancer p53 jodhpur pharmacy collage Despite the high number of previous studies, the role of p53 alterations in prostate cancer is not clearly defined. To address the role of p53 alterations in prostate cancer biology, a total of 2514 cancers treated by radical prostatectomy were successfully analyzed by immunohistochemistry in a tissue microarray format. Overall a low rate of p53-positive tumors was found (2.5%). A significant underestimation of p53-positive cases was excluded by subsequent large section analyses and direct sequencing of the p53 gene in subsets of our patients. Large section analysis of 23 cases considered negative on the tissue microarray yielded only one weakly p53-positive tumor. Only 4 out of 64 (6.4%) high-grade tumors, that were considered negative for p53 by immunohistochemistry, presented exon 5–8 mutations. Conclusion These data suggest a high sensitivity of our immunohistochemistry approach and confirm the overall low frequency of p53 alterations in clinically localized prostate cancer. A positive p53 immunostaining was strongly associated with presence of exon 5–8 mutations (P<0.0001), advanced pT-stage (P<0.0001), high Gleason grade (P<0.0001), positive surgical margins (P=0.03) and early biochemical tumor recurrence (P<0.0001). A higher rate of positive p53 immunostaining was detected in late-stage diseases including metastatic prostate cancer (P=0.0152) and hormone-refractory tumors (P=0.0003). Moreover, p53 expression was identified as an independent predictor of biochemical tumor recurrence in the subgroup of low- and intermediate-grade cancers. In summary, the results of this study show that p53 mutations characterize a small biologically aggressive subgroup of prostate cancers with a high risk of progression after prostatectomy. The rate of p53 alterations increases with prostate cancer progression. [97] Page 43 of 62
    • Cancer p53 jodhpur pharmacy collage 8.Future trends of p53 in curing various cancers 8.1 Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-κB pathways The p53 and nuclear factor-κB (NF-κB) pathways play crucial roles in human cancer, in which inactivation of p53 and hyperactivation of NF-κB is a common occurrence. Activation of p53 and inhibition of NF-κB promotes apoptosis. Although drugs are being designed to selectively activate p53 or inhibit NF-κB, there is no concerted effort yet to deliberately make drugs that can simultaneously do both. Recent results suggest that a surprising selection of small molecules have this desirable dual activity. In this Review we describe the principles behind such dual activities, describe the current candidate molecules and suggest mechanisms and approaches to their further development.[99] 8.2 Novel Regulatory Mechanism Identified for Key Tumor Suppressor p53: (Philadelphia – November 15, 2006) – Collaborating scientists from The Wistar Institute in Philadelphia and The Vienna Biocenter in Austria identified a novel mechanism involved in normal repression of the p53 protein, perhaps the single most important molecule for the control of cancer in humans. The new molecular pathway described in the study suggests intriguing approaches to diagnosing or intervening in the progression of many types of cancer. A report on the team‘s findings will be published online November 15 in the journal Nature. Shelley L .Berber says that ―The p53 protein is vital for controlling cancer throughout the body,‖ the Hilary Koprowski Professor said that at The Wistar Institute and senior author on the study. ―The new mechanism we describe, driven by a previously unknown enzyme, represses the p53 protein when its activity is not needed. Page 44 of 62
    • Cancer p53 jodhpur pharmacy collage ―What we‘re looking at now is the possibility that this enzyme, if over-expressed or over- active, might interfere with p53‘s normal tumor suppressor function and perhaps cause cancer. If that‘s the case, then we could develop drugs to inhibit the enzyme that would have the effect of freeing p53 to do its job of suppressing cancer. Unusually high levels of the newly identified enzyme might also be useful as a diagnostic marker for cancer.‖ Responsible for tumor suppression throughout the body, the p53 protein has been found to be mutated and dysfunctional in more than half of human cancers. When working properly, p53 acts by binding to DNA to activate genes that direct cells with damaged DNA to cease dividing until the damage can be repaired. Cells with such damage include cancer cells, since all cancers track to genetic flaws of one kind or another, whether inherited or acquired. If repairs cannot be made, p53 commands the cells with damaged DNA to self- destruct so they are no longer a danger to the body. This powerful ability of the p53 protein to shut down cell division and induce cell death points to why the availability of a repressive mechanism such as the one outlined in the new study might be crucial for cellular survival. Conclusion In their study, the scientists identified an enzyme called Smyd2 that adds a methyl group to the p53 protein at a specific site, with the result being that p53 cannot bind to DNA and, therefore, cannot act. The ability to bind to DNA is critical for p53‘s function,‖ says Jing Huang, Ph.D., one of the study‘s two lead authors. ―What we found was that methylation at the site we identified prevents p53 from binding to DNA, which also explains why it‘s a repressive modification.Berger and Huang note that this is one of only a small number of studies to identify methylation as playing a role in regulating the activity of proteins that are not histones. Histones are relatively small proteins around which DNA is coiled to create structures called nucleosomes. Compact strings of nucleosomes, then, form into chromatin, the substructure of chromosomes.With histones, methylation is well recognized as a regulatory mechanism, but the fact that other proteins are also be modified in the same way is a relatively new observation. Berger believes that scientists will likely find this type of regulatory mechanism at work in many other protein systems over the next few years. Page 45 of 62
    • Cancer p53 jodhpur pharmacy collage Interestingly, only one other study has shown a role for methylation in regulating p53. In that study, a methyl group added to a specific site on p53 called K372 was shown to activate the tumor-suppressor molecule rather than repress it. The site identified in the current study, dubbed K370, is adjacent to that first site. An additional finding of note is that the two sites interact closely. Huang says that there‘s important crosstalk between the two sites, but only in one direction. If the previously identified site is already methylated, the site we found cannot be methylated. But the reverse is not the case. The two lead authors on the Nature study are Jing Huang, Ph.D., at Wistar and Laura Perez-Burgos, Ph.D., at The Vienna Biocenter. The additional Wistar co-authors are Brandon J. Placek, Ph.D., Jean A. Dorsey, and senior author Shelley L. Berger, Ph.D. The additional co-authors at The Vienna Biocenter are Roopsha Sengupta, Mario Richter, Stefan Kubicek, and Thomas Jenuwein, a senior scientist who led the collaboration at The Vienna Biocenter. Funding for the research was provided to the Berger laboratory by the National Institutes of Health and the Commonwealth Universal Research Enhancement Program of the Pennsylvania Department of Health. Support for the Jenuwein laboratory was provided by Boehringer Ingelheim, the European Union, and the Austrian Ministry of Education, Science, and Culture. The Wistar Institute is an international leader in biomedical research, with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the country, Wistar has long held the prestigious Cancer Center designation from the National Cancer Institute. Discoveries at Wistar have led to the creation of the rubella vaccine that eradicated the disease in the U.S., rabies vaccines used worldwide, and a new rotavirus vaccine approved in 2006. Wistar scientists have also identified many cancer genes and developed monoclonal antibodies and other important research tools. Today, Wistar is home to eminent melanoma researchers and pioneering scientists working on experimental vaccines against flu, HIV, and other diseases. The Institute works actively to transfer its inventions to the commercial sector to ensure that research advances move from the laboratory to the clinic as quickly as possible. [100] Page 46 of 62
    • Cancer p53 jodhpur pharmacy collage 8.3 p53 at the cross roads of cancer and ageing ABSTRACT The p53 tumour suppressor plays an undisputed role in cancer. p53's tumour suppressive activity stems from its ability to respond to a variety of stresses to trigger cell cycle arrest, apoptosis or senescence, thereby protecting against malignant transformation. An increasing body of evidence suggests that p53 also drives organismal ageing. Although genetic models with altered p53 function display age-related phenotypes and thus provide in vivo evidence that p53 contributes to the ageing process, p53's role in organismal ageing remains controversial. Anti-cancer therapies that target p53 and reactivate or enhance its activity are considered good alternatives for treating various neoplasms. Therefore, it is important to determine whether these clinical approaches compromise tissue homeostasis and contribute to ageing. This review presents a number of models with altered p53 function and discusses how these models implicate p53 as part of a molecular network that integrates tumour suppression and ageing. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.[101] 8.4 p53 statuses may be predictive of outcome in breast cancer The association between p53 alterations and clinical outcome in breast cancer has been the subject of numerous investigations. The possibility that p53 status influences biological behaviour was raised in an early study in which the presence of p53 mutations in aggressive breast cancer was demonstrated [37], and the majority of studies support an association between worse survival and the presence of p53 mutations. This association was confirmed in a comprehensive meta-analysis of the effect of somatic p53 mutations on prognosis in breast cancer [8]. Potential correlations between the type of p53 mutation and the clinical phenotype in breast cancer have been described [38]. In this study, it was shown that mutations affecting amino acids critical for DNA binding were associated with very aggressive cancers, whereas null mutations and other missense mutations were associated with an Page 47 of 62
    • Cancer p53 jodhpur pharmacy collage indeterminate clinical phenotype. A recent study suggests that p53 mutation may be an important molecular genetics correlate of breast cancer progression [39]. In a further study of primary breast carcinomas, expression of the angiogenic vascular endothelial growth factor was shown to correlate with poor prognosis and with mutation in p53 [40]. The serpin family member maspin is an inhibitor of angiogenesis, invasion and metastasis. A step-wise decrease in the expression of maspin in the sequence DCIS > invasive cancer > lymph node metastasis has been described, strongly supporting an important role in breast cancer progression [41]. Maspin is directly transcriptionally induced by wild-type p53, thus providing an interesting connection between p53 and progression in ductal breast carcinomas [42]. It will clearly be of interest to determine how expression of maspin relates to p53 status in breast cancer. Studies of the effect of p53 mutations on chemo sensitivity of human tumours have produced conflicting results. In breast cancer, there is evidence that specific mutations correlate with primary resistance to doxorubicin and that the presence of such mutations may be predictive of early relapse [43]. This hypothesis was further supported by a later study from the same group [44]. In another study, cancers with p53 mutation were more likely to respond to paclitaxel [45]. A number of recent reports have described the detection of tumour-specific DNA in plasma from patients with breast carcinomas. p53 mutations can be detected in peripheral blood in a significant proportion of patients whose primary tumours contain mutations. Furthermore, the presence of p53 mutations in plasma DNA is strongly correlated with various clinicopathological parameters and is a significant prognostic factor [46]. p53 autoantibodies are also detectable in patients with breast cancer. These were reported to occur in 15% of patients but the presence of such antibodies had no relationship to disease status [47].[102] 8.5 Suggested therapies on combination and improvement of p53 therapy  cisplatin based chemotherapy  Avoidance of estrogen foodstuff – eg. soy Page 48 of 62
    • Cancer p53 jodhpur pharmacy collage  Consumption of more alkaline foodstuff – as cancer survives and multiplies in acidic environment.  Use of herbal medicine like – tulsi [osmium sanctum], Granoda lucidum has been a revolutionary fungal mushroom having high anti neoplastic activity .  Use of antioxidant vitamins A .D .E etc as cancer needs oxygen for its survival .  Use of stem therapy is also tried on experimental basis .  Monoclonal antibody therapy.  Human pappillavirus vaccines.  Vectors enhance the p53 cancer gene therapy  Viral vectors – lentivirus, adenovirus ONYX -015, herpes, retrovirus act as a carrier of p53 gene in cancer tumor which undergo apoptosis .  Non viral vectors –gene gun, lipofaction nakedplasmids, RNA, c ationic liposome peptide DNA complex are carrier of this gene therapy.  now the use of virus with combination of liposomes complex is used on animal xenograft models[103] 8.6 p53 and stem cells: new developments and new concerns. As the guardian of the genome, the tumor suppressor p53 prevents the accumulation of genetic mutations by inducing cell cycle arrest, apoptosis or senescence of somatic cells after genotoxic and oncogenic stresses. Recent studies have identified the roles of p53 in suppressing pluripotency and cellular dedifferentiation. In this context, p53 suppresses the self-renewal of embryonic stem cells after DNA damage and blocks the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). If the inactivation of p53 pathway is a prerequisite for successful reprogramming, these findings raise concerns for the genomic stability and tumorigenecity of iPSCs and their derivatives. Elucidation of the roles of p53 as a barrier to pluripotency and cellular dedifferentiation might also reveal the mechanisms by which p53 coordinates tumor suppression and aging. [104] Page 49 of 62
    • Cancer p53 jodhpur pharmacy collage 8.7 Inhibition of p53 Transcriptional Activity: A Potential Target for Future Development of Primary Demyelination Oligodendrogliopathy, microglial infiltration, and lack of remyelination are detected in the brains of patients with multiple sclerosis and are accompanied by high levels of the transcription factor p53. In this study, we used the cuprizone model of demyelination, characterized by oligodendrogliopathy and microglial infiltration, to define the effect of p53 inhibition. Myelin preservation, decreased microglial recruitment, and gene expression were observed in mice lacking p53 or receiving systemic administration of the p53 inhibitor pifithrin- , compared with untreated controls. Decreased levels of lypopolysaccharide-induced gene expression were also observed in vitro, in p53–/– primary microglial cultures or in pifithrin- -treated microglial BV2 cells. An additional beneficial effect of lack or inhibition of p53 was observed in Sox2+ multipotential progenitors of the subventricular zone that responded with increased proliferation and oligodendrogliogenesis. Based on these results, we propose transient inhibition of p53 as a potential therapeutic target for demyelinating conditions primarily characterized by oligodendrogliopathy. Multiple sclerosis (MS) is a demyelinating disorder characterized by heterogeneity of clinical and neuropathological signs and disease susceptibility (Lucchinetti et al., 2000 ). At least two broad categories have been defined based on neuropathological findings: one is characterized by immune-mediated demyelination occurring at perivenous locations, whereas the other one is characterized primarily by myelin loss consequent to oligodendrogliopathy and is associated with extensive microglial infiltration (Barnett and Prineas, 2004 ). An additional difference between these two neuropathological findings is the presence of remyelination attempts that are frequently detected in the immune- mediated forms and are less prominent in cases with extensive oligodendrogliopathy (Lucchinetti et al., 2000 ). Very little is known regarding the causes of oligodendroglial death in MS patients; however, increased levels of the pro-apoptotic transcription factor p53 and of its downstream genes have been detected in cases characterized by oligodendrogliopathy, microglial infiltration, and relative lack of remyelination (Kuhlmann et al., 2002 ; Aboul-Enein et al., 2003 ; Wosik et al., 2003 ; Stadelmann et al., 2005 ). Page 50 of 62
    • Cancer p53 jodhpur pharmacy collage Studies in cultured human oligodendrocytes further identified p53 as a critical pro- apoptotic effector (Ladiwala et al., 1999 ; Wosik et al., 2003 ). We thereby hypothesized that this transcription factor could play a very important role in models of primary demyelination consequent to oligodendrocyte dystrophy. To test this hypothesis, the cuprizone model of toxic demyelination has been adopted , which is characterized by oligodendrocyte apoptosis and microglial infiltration (Hiremath et al., 1998 ; Matsushima and Morell, 2001 ).[105] 8.8 Gene Therapy for Pediatric Cancer: State of the Art and Future Perspectives While modern treatments have led to a dramatic improvement in survival for pediatric malignancy, toxicities are high and a significant proportion of patients remain resistant. Gene transfer offers the prospect of highly specific therapies for childhood cancer. ―Corrective‖ genes may be transferred to overcome the genetic abnormalities present in the precancerous cell. Alternatively, genes can be introduced to render the malignant cell sensitive to therapeutic drugs. The tumor can also be attacked by decreasing its blood supply with genes that inhibit vascular growth. Another possible approach is to modify normal tissues with genes that make them more resistant to conventional drugs and/or radiation, thereby increasing the therapeutic index. Finally, it may be possible to attack the tumor indirectly by using genes that modify the behavior of the immune system, either by making the tumor more immunogenic, or by rendering host effector cells more efficient. Several gene therapy applications have already been reported for pediatric cancer patients in preliminary Phase 1 studies. Although no major clinical success has yet been achieved, improvements in gene delivery technologies and a better understanding of mechanisms of tumor progression and immune escape have opened new perspectives for the cure of pediatric cancer by combining gene therapy with standard therapeutic available treatments. [106] 9.SUMMARY AND CONCLUSION Page 51 of 62
    • Cancer p53 jodhpur pharmacy collage p53 was originally viewed as an oncogene,but during the past several decades it has come to be understood to be a tumor suppressor gene.During this time , many p53 family transcriptional targets have been identified as having the capacity to modulate various cellular processes including growth arrest , apoptosis , senescence , differentiation , and DNA repair. In fact, it has become evident that this small 53-kDa tumor suppressor is a molecular node at the crossroads of an extensive and complex network of stress responsive pathways . Deregulation of p53 has enormous influence on carcinogenesis as mt p53 can induce an increased epigenetic instability of tumor cells, facilitating and accelerating the evolution of the tumor. Understanding the mechanisms of p53‘s function is currently a major challenge in p53 research, and such knowledge may ultimately provide novel targets and approachs to therapeutic manipulation of the p53 pathway in the treatment of cancer. The challenge in the future will be to use our knowledge of p53 to develop more highly effective strategies and novel drugs for cancer prevention and treatment with fewer side effects. As chemotherapy show disadvantage of regrowth of cell , metastasis after surgery treatment , chemoresistance to tumor, gene therapy has been a revolutionary step towards cancer treatment . Genetic correction strategies are presently being developed and tested in animal models for human malignancies and in early patient trials. The cancer susceptibility genes p53 have been tested in ovarian & breast cancer patients, , and have shown some potential for this antitumor strategy. P53 gene therapy may be effective even against tumors that lack p53 mutations, because p53 may function as a growth inhibitor in a variety of gene transfer settings. Key problems at present include the degradation of vector by the immune system and a need for higher levels of gene transduction. Solutions will require the development of improved vectors, improved vector delivery systems, and the fine-tuning of human gene therapy in appropriate models of human cancer. This small pocket size molecule has yet to see a lot of changes means sky is not the limit and the end of land on earth might also not be its end in coming future.This molecule being crowned also as the molecule of the year in 1999 promises that it will combat cancer by all its powers of apoptosis and cell death only on the condition if people on earth stop smoking there golden lives with a small ―FOUR SQUARE‖ and let mutations happen in p53.So lets stop being exposed to harmful rays and be away from smoking and more near life.Rest work the intelligent p53 will do on its own. Page 52 of 62
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