Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells.

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.     

deltaNp73 facilitates cell immortalization and cooperates with oncogenic Ras in cellular transformation in vivo

TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, DeltaNp73, is upregulated in breast and gynecological cancers. We further showed that DeltaNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of DeltaNp73, this can only be directly shown in primary cells. We report here that DeltaNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. DeltaNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, DeltaNp73 rescues Ras-induced senescence. Moreover, DeltaNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of DeltaNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of DeltaNp73. Taken together, DeltaNp73 behaves as an oncogene that targets p53 that might explain why DeltaNp73 upregulation may be selected for during tumorigenesis of human cancers.     

Role of p73 in malignancy: tumor suppressor or oncogene?

The recently identified p53 family member, p73, shows substantial structural and functional homology with p53. However, despite the established role of p53 as a proto-type tumor suppressor, a similar function of p73 in malignancy is questionable. Overexpression of p73 can activate typical p53-responsive genes, and activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human tumors, led to the hypothesis that p73 has tumor suppressor activity just like p53. However, unlike p53-/- mice, p73 knockout mice do not develop tumors. Extensive studies on primary tumor tissues have revealed overexpression of wild-type p73 in the absence of p73 mutations instead, suggesting that p73 may augment, rather than inhibit tumor development. In contrast to p53, differential splicing of the TP73 gene locus gives rise to a complex pattern of interacting p73 isoforms with antagonistic functions. In fact, induction of apoptosis by increased levels of p73 can be blocked by both p53 mutants and the N-terminally truncated p73 isoforms, which were recently shown to possess oncogenic potential. In the light of these new findings the contradictory role of p73 in malignancy will be discussed.     

Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells

Aberrations in centrosome numbers have long been implicated in aneuploidy and tumorigenesis, but their origins are unknown. Here we have examined how overexpression of Aurora-A kinase causes centrosome amplification in cultured cells. We show that excess Aurora-A does not deregulate centrosome duplication but gives rise to extra centrosomes through defects in cell division and consequent tetraploidization. Over expression of other mitotic kinases (Polo-like kinase 1 and Aurora-B) also causes multinucleation and concomitant increases in centrosome numbers. Absence of a p53 checkpoint exacerbates this phenotype, providing a plausible explanation for the centrosome amplification typical of p53-/- cells. We propose that errors during cell division, combined with the inability to detect the resulting hyperploidy, constitute a major cause for numerical centrosome aberrations in tumors.     

Guilt by association? p53 and the development of aneuploidy in cancer

Aneuploidy is one of the most frequent genetic alterations in solid tumors. It is commonly caused by cell division errors that are induced by oncogene activation or loss of tumor suppressor functions. In addition, certain viral oncoproteins have been implicated in the induction of chromosome copy number changes. Aneuploidy and inactivation of p53 frequently coincide in human cancers but there is increasing evidence that loss of p53 by itself is not a primary cause of aneuploidy. Nonetheless, p53 inactivation synergizes with additional oncogenic events to promote aneuploidy and may facilitate chromosomal imbalances through indirect mechanisms. This review summarizes the current knowledge about the association between aneuploidy and p53, and discusses two of the most controversial mechanisms that have been implicated in genomic instability associated with loss of p53: subversion of ploidy control and aberrant centrosome duplication.     

p53-based Cancer Therapy

Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNA''s that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues.Thirty years of research on p53 have produced a detailed understanding of its structure and function. The almost universal loss of p53 activity in tumors has spurred an enormous effort to develop new cancer treatments based on this fact. Sophisticated animal models have shown that activation of the p53 response in even advanced tumors can be curative (Martins et al. 2006; Ventura et al. 2007; Xue et al. 2007). The p53 gene therapy, Gendicine, is approved in China and its US counterpart, Advexin, has shown activity in number of clinical trials. The p53 protein level is raised in many tumors by virtue of an increase in the protein''s half life and this tumor specific alteration in p53 processing has attracted tumor immunologists, who are now testing a number of p53 based vaccines in cancer patients (Speetjens et al. 2009).In more conventional approaches a range of small druglike molecules targeting the p53 system have been developed and several are now in clinical trials. Of critical importance has been the development of small-molecule inhibitors of the p53–Mdm2 protein interaction such as the Nutlins (Vassilev et al. 2004), which have shown activity against human xenografts in preclinical models. Advanced structural approaches have provided compelling support for the idea that some mutant p53 proteins can be targets for small molecules that would cause them to regain wild-type function (Joerger et al. 2006). Cell based screening methods have identified small molecules that can activate both mutant and wild-type p53 proteins in tumor cells to induce apoptosis. These screens, and RNAi based approaches, have revealed many new targets for therapy in the p53 pathway. In an exciting new approach, that has been validated in other tumor suppressor pathways, the search is on for targets in pathways that will show synthetic lethal interactions with loss of p53 function. Finally drug combinations have been developed that can selectively kill cancer cells that lack p53 function while protecting normal cells (Sur et al. 2009). The next few years hold out the prospect of new p53 based therapies that will be of wide application in cancer and other diseases.     

Effect of p53 on centrosome amplification in prostate cancer cells.

Chromosomal instability (CIN) is one of the common features in prostate cancer, especially in advanced stages. Recently, the involvement of p53 in CIN through the regulation of centrosome amplification has been proposed in certain tumor types. In this study, we investigated the relationship between p53 and centrosome amplification in prostate cancer cells. Increased centrosome number and size were observed in DU145 and PC3 containing nonfunctional p53 compared to LNCap which expressed wild-type p53. Transfection of p53 into PC3 cells resulted in a decreased cell growth rate, G2/M arrest and decreased centrosome abnormalities. We provide the first evidence on a correlation between loss of p53 function and centrosome amplification in prostate cancer cells. Our results indicate that p53 may play a role in the regulation of centrosome amplification and loss of p53 may be one of the mechanisms involving CIN in prostate cancer cells.     

Wild-type p53: tumors can't stand it

Most malignant tumors disrupt the p53 signaling pathway in order to grow and survive. Although many genes in addition to p53 are mutated in tumors, recent studies by Ventura et al. (2007) and Xue et al. (2007) suggest that restoring p53 function alone is sufficient to cause regression of several different tumor types in mice and thus might represent a potent therapeutic strategy to treat certain human cancers. Martins et al. (2006) also demonstrate that restoration of p53 activity results in tumor regression but add the sobering caveat that tumors may be able to quickly generate resistance by finding other ways to disrupt the p53 pathway.     

ECRG2 disruption leads to centrosome amplification and spindle checkpoint defects contributing chromosome instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.     

Mutant p53 Gain-of-Function in Cancer

In its wild-type form, p53 is a major tumor suppressor whose function is critical for protection against cancer. Many human tumors carry missense mutations in the TP53 gene, encoding p53. Typically, the affected tumor cells accumulate excessive amounts of the mutant p53 protein. Various lines of evidence indicate that, in addition to abrogating the tumor suppressor functions of wild-type p53, the common types of cancer-associated p53 mutations also endow the mutant protein with new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Collectively, these activities are referred to as mutant p53 gain-of-function. This article addresses the biological manifestations of mutant p53 gain-of-function, the underlying molecular mechanisms, and their possible clinical implications.Mutations in the TP53 gene, encoding the p53 tumor suppressor, are arguably the most frequent type of gene-specific alterations in human cancer. This attests to the centrality of p53 as a major mainstay in the body’s built-in anticancer defense mechanisms. Not surprisingly, this pivotal role of the wild-type p53 (wtp53) protein in tumor suppression has attracted many researchers to study it in detail, resulting in an avalanche of information and publications. One might expect that, similar to other tumor suppressor genes, the sole outcome of mutations in the TP53 gene will be loss of wtp53 function, characteristically manifested as total lack of p53 expression or production of unstable or truncated mutant proteins. Yet, quite strikingly, the vast majority of cancer-associated p53 mutations actually lead to production of full length protein, typically with only a single amino acid substitution, which tends to accumulate in the tumor cells and reach steady-state levels that greatly exceed those of wtp53 in noncancerous cells (Rotter 1983). This remarkable feature has suggested early on in p53 research that cancer-associated mutant p53 (mutp53) isoforms may be more than just relics of wtp53 inactivation, and may instead play distinctive roles in the tumor cells.In principle, emergence of a p53 mutation within a cell might have three, not mutually exclusive, types of outcome (Michalovitz et al. 1991; Sigal and Rotter 2000; Weisz et al. 2007b). First, such mutation is expected to abrogate the tumor suppressor function of the affected TP53 allele, reducing the overall capacity of the cell to mount a proper p53 response; if both alleles eventually become mutated, or if the remaining allele is lost, such cells will be totally deprived of anticancer protection by p53. Second, many common mutp53 isoforms can exert dominant–negative effects over coexpressed wtp53, largely by forming mixed tetramers that are incapable of DNA binding and transactivation. Hence, even if one wt allele is retained, the cell may be rendered practically devoid of wtp53 function through such mechanism, particularly if the mutant protein is expressed in excess over its wt counterpart. Third, and most relevant for this article, the emergent mutp53 protein might possess activities of its own, often not present in the original wtp53 protein, which can actively contribute to various aspects of tumor progression. Such activities, commonly described as mutp53 gain-of-function (GOF), are the subject of this article. Several recent reviews address in detail the various aspects of mutp53 GOF (Brosh and Rotter 2009; Donzelli et al. 2008; Lozano 2007; Olivier et al. 2009; Peart and Prives 2006; Petitjean et al. 2007; Song and Xu 2007; Strano et al. 2007; Weisz et al. 2007b). Therefore, we focus here mainly on general principles as well as on some of the more recent findings.     

c-MYC, RB-1, TP53, and centromere 8 and 17 copy number in B-cell non-Hodgkin's lymphomas assessed by dual-color fluorescence in situ hybridization.

Dual-color interphase FISH was performed to B-cell Non-Hodgkin's lymphomas to detect numerical genetic alterations in c-MYC, RB-1, TP53 and centromere 8 and 17. The probe combinations c-MYC/centromere 8, RB-1/centromere 8 and TP53/centromere 17 were applied, and the hybridization signals scored in a correlated fashion. Copy number aberrations was found in 24 of 45 lymphomas examined (53%). Nine tumors (20%) had increased c-MYC gene copy number (three to 8 copies). Seven of these nine had increase in c-MYC copies relative to centromere 8 copy number. Allelic loss of RB-1 was found in 9 tumors (20%), one tumor lacked both alleles. Nine cases (20%) showed hemizygous TP53 deletion. TP53 deletion was significantly associated with high-grade histology (P = 0.02). Numerical alterations in c-MYC and RB-1 were not associated with prognosis. Patients with hemizygous TP53 deletion had shorter survival (relative risk = 6.1, P<0.001) than the ones with two or more alleles.     

Aneuploidy,centrosome activity and chromosome instability in cells deficient in homologous recombination repair

Chromosome instability and loss or gain of chromosomes are changes characteristic of many tumour cells and human disorders. However, the mechanism of these changes has not yet been fully determined. We have recently shown that hamster cell lines deficient in homologous recombination repair (HRR) genes XRCC2 and XRCC3 have an elevated frequency of aneuploidy compared with wild-type cells and mutant cells transfected with the appropriate human gene. In addition, XRCC2 and XRCC3 deficient hamster cell lines show a high frequency of multiple centrosomes and abnormal spindle formation. Cells deficient in HRR show a high frequency of both chromosome-type and chromatid-type aberrations, which could potentially lead to mis-segregation. The role of chromosome aberrations and other factors, including chromosome lagging, premature chromatid separation, and centrosome malfunctioning on chromosome mis-segregation in irs1 and irs1SF cells have been investigated. In particular, the linkage of DNA repair proteins with centrosomes suggests a key role for the centrosome in controlling cellular repair processes.     

TP53 codon 72 and intron 3 polymorphisms and mutational status in gastric cancer: an association with tumor onset and prognosis

Although TP53 alterations have been studied in human tumors, data considering the role of two common TP53 polymorphisms (Pro72Arg in codon 72 and Ins16bp in intron 3) and their associations with TP53 mutations in gastric cancer are very limited. Thus, we analyzed these parameters taking into consideration the clinicopathological data. DNA from 106 gastric tumor samples was available for TP53 Pro72Arg and TP53 Ins16bp polymorphism genotyping by PCR-RFLP and PCR, respectively. The mutational status of the TP53 exons 5-7 was assessed by the single-strand conformational polymorphism test. The TP53 72ArgArg genotype was statistically associated with patients aged ≥65 years (p = 0.039), and the intron 3 A2A2 genotype was correlated with late-stage tumors (III and IV; p = 0.043). Considering both polymorphisms, a negative correlation between the TP53 Pro-A1 haplotype and age <65 years (r = -0.211; p = 0.030) was found. Taking into account the TP53 mutations, the Pro/Pro genotype was positively correlated with the presence of exon 7 mutations (p = 0.049), and a correlation between this genotype and the number of mutations in TP53 was observed (p = 0.019). This study corroborates the understanding of TP53 polymorphisms in gastric carcinogenesis, especially regarding the genetic features in tumor onset and prognosis.     

Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line

The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the xrs strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronied populations of xrs cells, estimated to have been irradiated in late S and G2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.     

The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells

The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.     

A mitotic recombination in Wilms tumor occurs between the parathyroid hormone locus and 11p13

Summary Wilms tumor is believed to occur as the result of two mutations affecting both alleles of a critical gene located within the p13 band of chromosome 11 (Knudson and Strong 1972; Riccardi et al. 1978). Several mechanisms by which these mutations occur have already been determined in retinoblastoma (Cavenee et al. 1983) and Wilms tumor (Koufos et al. 1984; Orkin et al. 1984; Reeve et al. 1984; Fearon et al. 1984a; Eccles et al. 1984). Of the various mechanisms, however, no example of a mitotic recombination was demonstrated in Wilms tumor. An example is presented here which has been detected by the use of restriction fragment length polymorphisms (RFLPs) mapping to chromosome 11p. In addition the data presented are consistent with the mapping location of parathyroid hormone (PTH) being proximal to 11p13.     

p53 aberrations in low grade endometrioid carcinoma of the endometrium with nodal metastases: possible insights on pathogenesis discerned from immunohistochemistry

Background

TP53 mutations are rarely identified in low grade endometrioid carcinoma of the endometrium, and their pathogenic significance in such tumors is evidenced by the fact that TP53 aberrations have been associated with reduced recurrence-free survival in this subset of tumors. However, TP53 aberrations may not always represent a driving molecular event in a given endometrial cancer with a mutation. In this case study, the immunophenotype of a distinctive low grade endometrioid adenocarcinoma with an unusual pattern of lymph node metastases is used to explore the possible roles for underlying TP53-related molecular events in its pathogenesis.

Case presentation

A low grade endometrioid carcinoma, 9 cm in greatest dimension, with 35% invasion of the myometrial wall thickness, focal lymphovascular invasion, and metastases to 2 of 16 pelvic lymph nodes, was diagnosed in a 52-year-old woman. The endometrial tumor showed a p53-mutation (aberrant)-type immunohistochemical pattern in 40% of the tumor, but the rest of the tumor, as well as the foci of myometrial and lymphovascular invasion, were p53-wild type. Both lymph nodes with metastatic disease showed a distinct biphasic pattern, comprised of both p53-wild type and p53-aberrant areas in tumoral foci that were spatially apposed but not intermixed. Most p53-aberrant areas (at both the lymph nodes and the endometrium) showed a higher mitotic index and increased atypia as compared to the p53-wild type areas; both showed squamous differentiation. The p53-aberrant areas at both locations were also p16-diffusely positive, vimentin-positive, and estrogen/progesterone receptor-positive, whereas the p53-wild type areas showed an identical immunophenotype with the exception of being p16-mosaic positive. All components of the tumor at both the primary and metastatic sites showed loss of MSH2 and MSH6 and retained MLH/PMS2 expression.

Conclusions

The presence of p53-mutant and wild-type areas in multiple lymph nodes, coupled with the absence of a p53-aberrant immunophenotype in the myometrium-invasive or lymphovascular-invasive portions of the tumor, argues against the possibility that the TP53 mutation in this tumor is a driving event in its pathogenesis, at least regarding the metastatic process. This case illustrates how routine immunohistochemistry can provide important insights into underlying molecular events in cancers, exemplifies an uncommon co-existence of DNA mismatch repair protein deficiency and p53-aberrant immunophenotype in low-grade endometrioid carcinoma, illustrates morphologic differences between p53-aberrant and p53-wild type areas within in the same tumor, and is an exemplar of the emerging theory that lymph node metastases of endometrial cancer may be comprised of different subclones of the primary tumor.