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Journal of Stress Physiology & Biochemistry, Vol. 7 No. 3 2011, pp. 143-151 ISSN 1997-0838 Original Text Copyright © 2011 by Singh, Singh, Singh and Gambhir
REVIEW
Tumour Suppressor p53 and its prognostic value in mutation analyses in Breast Cancer patients
Singh Shinjini 1, Mritunjai Singh 1, Rakesh K. Singh 2, Indrajeet S. Gambhir 1#
1 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India
2 Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, 221
005, India *Email: i [email protected] Phone: +91-9415255998 Fax: +91-542-2307521
Received June 20, 2011
The tumour suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, apoptosis, DNA repair and angiogenesis in response to oncogenic or other cellular stress signals. According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through these interventions of many modulator proteins. In this review, we provide insights into the structural complexity of p53, with specific interest on its role in breast cancer. p53 emerges as a paradigm for a more general understanding of the structural organisation of modulator proteins and the effects of disease-causing mutations.
Key words: transcription factor, p53 gene, p53 protein, prognosis, breast cancer.
REVIEW
Tumour Suppressor p53 and its prognostic value in mutation analyses in Breast Cancer patients
Singh Shinjini 1, Mritunjai Singh 1, Rakesh K. Singh 2, Indrajeet S. Gambhir 1#
1 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India
2 Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, 221
005, India *Email: i [email protected] Phone: +91-9415255998 Fax: +91-542-2307521
Received June 20, 2011
The tumour suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, apoptosis, DNA repair and angiogenesis in response to oncogenic or other cellular stress signals. According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through these interventions of many modulator proteins. In this review, we provide insights into the structural complexity of p53, with specific interest on its role in breast cancer. p53 emerges as a paradigm for a more general understanding of the structural organisation of modulator proteins and the effects of disease-causing mutations.
Key words: transcription factor, p53 gene, p53 protein, prognosis, breast cancer.
p53 is essential for the prevention of cancer development. In response to oncogenic or other cellular stress, p53 is activated and induces up- or downregulation of a variety of genes involved in cell cycle arrest, DNA repair, senescence, or apoptosis (Vogelstein et al., 2000). The transcriptional activity of p53 is controlled by a complex feedbackregulated network via the negative-regulator MDM2 in concert with its homolog MDM4 (Brooks and Gu, 2006). It is further regulated through a multitude of post translational modifications and interaction with a variety of signalling proteins (Toledo and Wahl,
2006). p53 gene and its protein product have become the centre of intensive study ever since it became clear that more than 50% of human cancers harbour mutations in this gene (Arnold, 1997).
p53 Domains: Structure and function
The p53 protein is a transcriptional factor that enhances the rate of transcription of several known genes that carry out the p53-dependent functions in a cell. Table 1 shows the products of the genes transcriptionally activated by p53. The human p53 protein contains 393 amino acids and has been
divided structurally and functionally into four domains (Arnold, 1997). The N-terminal region consists of an intrinsically disordered transactivation domain (TAD) and a proline-rich region. It is followed by the central, folded DNA-binding core domain that is responsible for sequence DNA binding. This domain is connected to a short
tetramerization domain, via a flexible linker, that regulates the oligomerization state of p53 (Figure 1). At the C-terminus, p53 contains the regulatory domain. This natively unfolded region is rich in basic amino acids and binds DNA nonspecifically (Andreas and Alan, 2008).
1 TAD 61 PRR 94 p53C 292 325 TET 356 CT393
Figure 1: Full length p53 showing domains consisting of an N-terminal transactivation domain (TAD), followed by proline-rich region (PRR), the central DNA-binding domain (p53C), the tetramerization domain (TET), and the extreme C terminus (CT).
Table 1: Products of genes transcripationally activated by p53
p21 Inhibits several cyclin-cyclin-dependent kinases; binds cdk's, cyclins, and PCNA; arrest the cell cycle
MDM2 Product of an oncogene; inactivates p53-mediated transcription and so forms an autoregulatory loop with p53 activity
GADD45 Induced upon DNA damage; binds to PCNA and can arrest the cell cycle; involved in DNA nucleotide excision repair
Cyclin G A novel cyclin
Bax A member of BCl2 family that promotes apoptosis
N-Terminal Region
It is natively unfolded and consists of an acidic TAD, which is subdivided into two sub domains TAD1 (residues 1-40) and TAD2 (residues 40-61), and a proline-rich region (residues 64-92) (Walker and Levine, 1996). The TAD is a promiscuous binding site for a number of interacting proteins, such as components of transactivation machinery, transcriptional co activators p300/CBP (Creb Binding Protein) and the negative regulators MDM2/MDM4 (Marine and Jochemsen, 2005). Due to binding of the p53 TAD to partner proteins, regions of nascent helical structure, present in the native state rigidify and become fully folded. A fragment comprising residues 15-29, adopts an a-helical conformation upon binding to a hydrophobic cleft in the N-terminal of MDM2 and MDM4
(Popowicz et al., 2007). The MDM2 binding region of the p53 TAD overlaps with parts of the binding site for the transcriptional function of p53 (Andreas and Alan, 2008).
Proline Rich Region
The proline-rich region that links the TAD to the DNA-binding domain in human p53 contains PXXP motifs (Walker and Levine, 1996). These motifs are not conserved among mammalian p53s, but the prevalence of pralines in this region is maintained, indicating a functional or structural requirement for a certain degree of rigidity (Toledo et al., 2007). The exact role of proline-rich region is poorly understood (Andreas and Alan, 2008).
DNA Binding Domain
p53 consists of an immunoglobulin-like P-
sandwich that provides the basic scaffold for the DNA-binding surface. The surface is subdivided into two structural motifs that bind to the minor groove and major groove of target DNA, respectively. The loop-sheet-helix motif, which docks to the DNA major groove, includes loop L1, P-strands S2 ans S2’, parts of the extended P-strand S10, and the C-terminal helix. The other half of the
DNA-binding surface is formed by two large loops, namely, L2 and L3, which are stabilized by a zinc ion (Figure 2) (Duan and Nilsson, 2006). Human p53 is only marginally stable and melts at slightly above body temperature, 440 - 450C, which dictates the stability of the full length protein (Ang et al, 2006).
Figure 2: Structure of DNA-free p53C showing loops L1, L2 and L3 alongwith the zinc ion.
Tetramerization Domain
Full length p53 reversibly forms tetramers via a tetramerization domain in the C-terminal region of the protein from residues 325 to 356 (Veprintsev et al., 2006). It can be very well described as a dimer of primary dimers (Figure 3). The monomeric tetramerization domain consists of a short P- strand
and an a- helix linked by a sharp turn. These two monomers form a primary dimer, which is stabilized via an anti parallel intermolecular ß- sheet and antiparallel helix packing with three residues (Leu-330, Ile-332, and Phe-341) forming the central hydrophobic core of this dimer. Two such dimmers associate through their helices to form a four-helix bundle tetramer (Mateu and Fersht, 1999).
Figure 3: p53 tetramerization domain as a dimmer of dimmers.
p53 in Breast Cancer
Indeed, p53 mutation is the most common genetic abnormality found in human cancer (Greenblatt et al., 1994). p53 mutation or alteration is observed in up to 50% of human cancers (Hollstein et al., 1994), including breast cancer where loss of p53 wild-type activity has been postulated as an important factor in the development of the disease (Ziyaie et al., 2000).
The human chromosomal location of p53 gene is 17 p 13.1. It encompasses 20 kb of DNA with 11 exons which on transcription gives a 3.0 kb of mRNA having 1179 open reading frame. On translation, this mRNA produces a 53 kDa protein (hence named p53) (GEORGE, 2011), which has already been described.
p53 mutations are common in breast cancer and have been reported at a rate of 15-50%, depending upon the stage of disease and the method of detection (Tsuda et al., 1993; Davidoff et al., 1991; Thor et al., 1992; Runnebaum et al., 1991; Bartek et al., 1990; Mazars et al., 1992). Inheritance of a p53 mutant allele results in a rare familial autosomal disorder, the Li-Fraumeni syndrome. It is characterized by a high incidence of multiple early cancers, including breast tumours (Lacroix et al., 2006). They have been found in most types of tumours, with frequencies ranging from 50% (cervix) to 50% (lung). Between 20 to 35% of breast tumours have been shown to express a mutant p53. However, most of the information on p53 mutations is derived from sequence analysis that included only exon 5 to 8 (residues 126 to 306) within TP53, and examination of the whole p53 coding sequence is beginning to reveal an increasing number of mutations in the N- and C- termini of the protein (Vousden and Lu, 2002). Nevertheless, majority of p53 mutations appear to be localized in the DNA
binding domain, in the central part of p53. In cells expressing a mutant p53, this protein is generally no longer able to control cell proliferation, which results in insufficient DNA repair and genetic instability (Lacroix et al., 2006).
The reported frequency of p53 alterations is dependent on the method of detection. Immunohistochemical staining (IHC) is a protein based method that is used commonly to detect alterations in the p53 because it is relatively inexpensive and easy to perform, especially on a large number of tumours. A mutation results in a prolonged protein half-life and accumulation of the protein in the nucleus (Davidoff et al., 1991). IHC detects this abnormal accumulation and is therefore thought to be an indirect indication of a mutation. 30-50% of breast tumours have accumulation of p53 protein as measured by IHC (Davidoff et al., 1991; Allred et al., 1993).
The optimal analysis of the p53 gene as a mutagen test requires careful attention to patient ascertainment biases, methods of sample processing and mutation detection (Table 2) with the exception of population-based ascertainment, a number of breast cancer studies have utilised optimal or nearly optimal methodology for this purpose (Hartmann et al., 1997). For example, DNA is amplified utilising either touch preparations (Kovach et al., 1991) or tissue sections from consecutive tumours with good clinical data, and genomic DNA is analyzed in all coding regions (are atleast exons 5 to 8), by direct sequencing or a screening method in which virtually 100% sensitivity had been demonstrated by blind analyses (Hartmann et al., 1996). When comparing mutational patterns, it is critical to compare mutations from identical exons because the patterns vary by exon especially when mutations inside and outside of exons 5-8 are compared (Hartmann et al., 1995).
Table 2: Methodological issues in mutation analysis
Methods Comments
Sampling 1. Population-based with clinical data 2. Consecutive tumours with clinical data 3. Larger tumours in which excess material is available Ideal Generally more feasible Least desirable due to bias in sampling
DNA isolation From clusters in touch preps From tissue section in tumour-rich regions (often microdissection is necessary) Gross tumour tissue Removes essentially all fibrous and inflammatory infiltrates; negligible amount of tumour required; heterozygous and hemizygous mutations are readily distinguished Problematic for tumours with significant inflammatory or fibrous infiltrates; distinguishing hemizygous and heterozygous mutations can be difficult Least desirable; many mutations will be missed
Nucleic acid analysis 1. Genomic DNA 2. cDNA Best, although occasional mutations not in the sequenced regions are missed Mutations sometimes destabilize RNA so heterozygous mutations can be missed
Regions analysed 1. All coding regions, splice junctions and regulatory regions 2. Exons 5-8 3. Individual exons Best Regions usually examined in breast cancer, 20% of mutations are outside of exons 5-8 Problematic; each exon has a different pattern of mutation, which reflects its biology
Mutation search Direct genomic sequencing SSCP Immunohistochemical detection of increased p53 expression Sequence in both directions is necessary to detect all heterozygous mutations The most widely used method; unpredictable sensitivity; 60-95% of mutations are detected Used by most early studies; false positives and false negatives are high
p53 gene mutations and prognosis and breast cancer
Therapeutic decisions are based on analyses of established clinical prognostic markers, such as axillary lymph node metastases, tumour stage, and estrogen/progesterone receptor status. Additional experimental prognostic markers included DNA-ploidy, S-phase fraction, HER2 expression (Gasparini and Harris, 1995). However, the established and experimental prognostic factors are of only moderate predictive value (Klijn et al., 1993).
More recently, a study examining the expression of p53 protein in a series of 136 primary breast carcinomas, 106 of which were analysed with a panel of four monoclonal antibodies. Some variability was found in the immunostaining depending on the antibody used. 17 tumour sections stained positively by IHC were analysed by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) for the presence of p53 mutation at the molecular level. 15 cases were found positive for mutation. Hence, the proportion of p53-stained cells does not seem to be
an exact representation of the number of cancer cells bearing a mutation within a tumour. A significant correlation was found between p53 expression, and grade III disease, estrogen or progesterone negativity, and HER2 positivity. According to this study there is a good overlap between the presence of mutation at the DNA level and positive immunostaining in the breast cancer since only 2/17 samples scored negative upon PCR-SSCP analysis. The correspondence might even be better given the fact that mutations may have been missed by PCR-SSCP. Therefore, the 2 tumours in which no mutation was detected despite the presence of positive staining many still bear a mutated p53 allele. If, according to it, positive immunostaining often corresponds to the presence of a mutation at the genetic level, the reverse is not always true (Jacquemier et al., 1994). Despite the fact that immunestaining does not permit the detection of all mutations affecting p53 (nonsense mutations do not lead to p53 accumulation), recent reports showing the possibility of p53 staining in the absence of detectable mutations are an additional argument in the favour of this method in the assessment of highrisk breast cancer patients.
The basis of negative associations between p53 mutations and prognosis is unclear. One analysis found evidence that the location of mutations within certain conserved regions of the gene is of especially poor prognostic significance (Bergh et al., 1995). The poor prognosis might be related to an increased proliferative capacity of tumour cells with mutant p53 protein and/or the greater resistance of such cells to growth inhibition by a variety of chemotherapeutic agents (Koechli et al., 1994).
Conclusions
p53 mutation patterns in various populations with high-risk for breast cancer differs significantly from those attributable to endogenous or background mutagenic processes. The multiple
“fingerprints” provide evidence that various mutagens can contribute to the development of breast cancer. However, additional data on the pattern of mutation in the p53 and other genes, and on the patient groups are necessary. With such data, comparison between the patterns of mutation in populations of the same genetic background living under different circumstances and in different ethnic groups living under comparable circumstances can be made. These studies will help in assessing the relative importance of environmental as opposed to endogenous events in the neoplastic process.
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