Wild-type P53 Protein Levels Research Articles

Early Social Enrichment Modulates Tumor Progression and p53 Expression in Adult Mice.

Epidemiological evidence indicates that stress and aversive psychological conditions can affect cancer progression, while well-being protects against it. Although a large set of studies have addressed the impact of stress on cancer, not much is known about the mechanisms that protect from cancer in healthy psychological conditions. C57BL/6J mouse pups were exposed to an environmental enrichment condition consisting of being raised until weaning by the biological lactating mother plus a non-lactating virgin female (LnL = Lactating and non-Lactating mothers). The Control group consisted of mice raised by a single lactating mother (L = Lactating). Four months after weaning, mice from LnL and L conditions were exposed to intramuscular injection of 3-methylcolantrene (3MCA), a potent tumorigenic drug, and onset and progression of 3MCA-induced fibrosarcomas were monitored over time. Pups from the LnL compared to the L group received more parental care and were more resilient to stressful events during the first week of life. In association, the onset of tumors in LnL adults was significantly delayed. At the molecular level, we observed increased levels of wild-type p53 protein in tumor samples of LnL compared to L adults and higher levels of its target p21 in healthy muscles of LnL mice compared to the L group, supporting the hypothesis of potential involvement of p53 in tumor development. Our study sustains the model that early life care protects against tumor susceptibility.

The correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in Surabaya

Background: Exposure of metals among dental technicians that come from the working environment can lead to the formation reactive oxygen species (ROS). ROS can cause mutations in the p53 gene (p53). The mutation is transversion mutation GuanineThymine. p53 mutations can lead to low expression of the wild-type p53 protein (p53). Wild-type p53 involved in many biological processes such as regulation of genes involved in cell cycle, cell growth after DNA damage, and apoptosis. However, exposure to metals among dental technicians can be prevented through the use of personal protective equipment (PPE) during work. Purpose: The purpose of this study was to analyze the correlation between the use of personal protective equipment to wild-type p53 protein levels among dental technicians in Surabaya. Method: This study was observational analytic with cross sectional approach. 40 samples were taken by random sampling. Data were retrieved through interviews and observations. Wild-type p53 was analyzed from saliva with indirect ELISA method. Analysis of data used Kolmogorov Smirnov normality test and a Pearson correlation test. Value significance was p<0.05 (95% confidence level). Result: There was a significant association between the use of personal protective equipment with wild-type p53 levels with p=0.002 Conclusion: The use PPE properly is positively correlated with the wild-type p53 protein levels of dental technicians in Surabaya.

Increased toll-like receptors and p53 levels regulate apoptosis and angiogenesis in non-muscle invasive bladder cancer: mechanism of action of P-MAPA biological response modifier.

BackgroundThe new modalities for treating patients with non-muscle invasive bladder cancer (NMIBC) for whom BCG (Bacillus Calmette-Guerin) has failed or is contraindicated are recently increasing due to the development of new drugs. Although agents like mitomycin C and BCG are routinely used, there is a need for more potent and/or less-toxic agents. In this scenario, a new perspective is represented by P-MAPA (Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride), developed by Farmabrasilis (non-profit research network). This study detailed and characterized the mechanisms of action of P-MAPA based on activation of mediators of Toll-like Receptors (TLRs) 2 and 4 signaling pathways and p53 in regulating angiogenesis and apoptosis in an animal model of NMIBC, as well as, compared these mechanisms with BCG treatment.ResultsOur results demonstrated the activation of the immune system by BCG (MyD88-dependent pathway) resulted in increased inflammatory cytokines. However, P-MAPA intravesical immunotherapy led to distinct activation of TLRs 2 and 4-mediated innate immune system, resulting in increased interferons signaling pathway (TRIF-dependent pathway), which was more effective in the NMIBC treatment. Interferon signaling pathway activation induced by P-MAPA led to increase of iNOS protein levels, resulting in apoptosis and histopathological recovery. Additionally, P-MAPA immunotherapy increased wild-type p53 protein levels. The increased wild-type p53 protein levels were fundamental to NO-induced apoptosis and the up-regulation of BAX. Furthermore, interferon signaling pathway induction and increased p53 protein levels by P-MAPA led to important antitumor effects, not only suppressing abnormal cell proliferation, but also by preventing continuous expansion of tumor mass through suppression of angiogenesis, which was characterized by decreased VEGF and increased endostatin protein levels.ConclusionsThus, P-MAPA immunotherapy could be considered an important therapeutic strategy for NMIBC, as well as, opens a new perspective for treatment of patients that are refractory or resistant to BCG intravesical therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2474-z) contains supplementary material, which is available to authorized users.

Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

BackgroundAdrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer.MethodsWe used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis.ResultsKnocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53.ConclusionsThese preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

Role of p53 in the progression of gastric cancer.

Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer (GC) but is poorly described in terms of molecular changes. Here, we explored the role of TP53, a commonly mutated gene in GC, to determine if p53 protein expression and/or the presence of somatic mutations in TP53 can be used as a predictive marker for patients at risk of progressing to GC from IM. Immunohistochemistry and high resolution melting were used to determine p53 protein expression and TP53 mutation status respectively in normal gastric mucosa, IM without concurrent GC (IM-GC), IM with concurrent GC (IM+GC) and GC. This comparative study revealed an incremental increase in p53 expression levels with progression of disease from normal mucosa, via an IM intermediate to GC. TP53 mutations however, were not detected in IM but occurred frequently in GC. Further, we identified increased protein expression of Mdm2/x, both powerful regulators of p53, in 100% of the IM+GC cohort with these samples also exhibiting high levels of wild-type p53 protein. Our data suggests that TP53 mutations occur late in gastric carcinogenesis contributing to the final transition to cancer. We also demonstrated involvement of Mdmx in GC.

Abstract 2226: Structure/function characterization of SBi4225, a novel inhibitor of the calcium-binding protein S100B.

Abstract The S100 family of proteins displays no enzymatic activity but has been shown to play roles in an array of biological functions such as calcium homeostasis, cellular growth, inflammation, and metabolism. The influence of these homodimeric proteins is mediated through protein-protein interactions in a typically calcium dependent manner and aberrant expression of S100 proteins has been linked to a variety of cancers, cardiomyopathies, and neurological disorders. Of particular interest, S100B is highly over-expressed in many cancers, including malignant melanoma (MM). In MM, S100B is used as prognostic indicator in evaluating treatment success and in predicting relapse. Studies now show that S100B is more than a clinical marker, and that it binds wild-type p53 in a calcium-dependent manner, sequestering it, and promoting its degradation. This results in the loss of p53-dependent tumor suppression activities. As inhibition of S100B expression by RNA interference restores both wild-type p53 protein levels and normal transcriptional activation/apoptosis activities, we have made it our long-term goal is to specifically inhibit the S100B-p53 interaction in order to restore the p53 tumor suppression function as a potential therapy for malignant melanoma. Although we have succeeded in identifying many S100B inhibitors (also known as SBiXs), optimization studies are a necessary part of the development of drugs with useful therapeutic activities. Included amongst these optimization studies are characterizations of structure-activity relationships (SAR). Our initial SAR studies have resulted in the development of a second series of SBiXs. Amongst this series is the novel S100B inhibitor, SBi4225. As part of the cyclical process of SAR studies, the binding of SBi4225 to S100B has been investigated using X-ray crystallography. As described within, SBi4225 binds to S100B in a position and orientation analogous to its predecessors, SBi4211 and SBi1. NMR data including chemical shift perturbations of 15N isotopically labeled protein backbone were consistent with the X-ray crystal structures and demonstrated the relevance of the SBi4225-S100B complex in solution. This new understanding of the SBi4225 structure/function relationship will undoubtedly impact the development and optimization of future SBiXs. Citation Format: Michael C. Cavalier, Laura McKnight, David Weber, Paul Wilder. Structure/function characterization of SBi4225, a novel inhibitor of the calcium-binding protein S100B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2226. doi:10.1158/1538-7445.AM2013-2226

Differential response between the p53 ubiquitin–protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

Pirh2, a recently identified ubiquitin–protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after γ irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human MCF-7 breast cancer cells

We reported recently that roscovitine arrested human MCF-7 cancer cells at G2-M phase of the cell cycle and concomitantly induced apoptosis. After roscovitine treatment, the level of wild-type p53 protein strongly increased and p53 was accumulated in the nucleus. Here, we raised the question of which pathway would be involved in roscovitine-induced apoptosis in MCF-7 cells, which are known to be caspase-3-deficient, and whether roscovitine-mediated activation of p53 protein might positively affect the execution of cell death. Roscovitine induced a depolarization of mitochondrial potential beginning at 6 hours posttreatment as evidenced by changes in J-aggregate formation and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Interestingly, roscovitine stimulated a site-specific phosphorylation of wild-type p53 protein in a time-dependent manner. p53 protein was specifically phosphorylated at Ser46. P-Ser46-activated wild-type p53 tumor suppressor up-regulated p53AIP1 protein, its downstream target known to mediate the depolarization of mitochondria. The onset of phosphorylation of p53 at Ser46 preceded the up-regulation of p53AIP1 protein and the depolarization of mitochondrial potential. We compared the kinetics of roscovitine-mediated p53 activation between caspase-3-deficient parental MCF-7 cells and cells reconstituted with caspase-3. The kinetics and the extent of p53 protein activation in caspase-3-proficient cells differed from those observed in caspase-3-deficient parental cells. Remarkably, roscovitine failed to induce phosphorylation at Ser46 in caspase-3-reconstituted MCF-7 cells. Our results indicate that, depending on the status of caspase-3 in MCF-7 cells, different apoptotic pathways were initialized.

Inhibiting S100B Restores p53 Levels in Primary Malignant Melanoma Cancer Cells

S100 calcium-binding proteins such as S100B are elevated in primary malignant melanoma and are used as markers for this and numerous other cancers. Wild-type p53 protein levels are relatively low in these cancer cells (i.e. when compared with cells without S100B) but are elevated when RNA antisense to S100B is introduced. This result implicates S100B in the down-regulation of p53 and is consistent with the large decreases in p53 protein levels observed previously in transient co-transfections of p53 and S100B (Lin, J., Blake, M., Tang, C., Zimmer, D., Rustandi, R. R., Weber, D. J., and Carrier, F. (2001) J. Biol. Chem. 276, 35037-35041). Down-regulation of p53 in primary malignant melanoma cells is likely the result of a direct interaction with S100B, which was observed by co-immunoprecipitation experiments. Furthermore, p53 binds regions of the S100B promoter, one of which matches the 20-nucleotide p53-binding consensus DNA sequence perfectly. Therefore, when p53 levels increase, it contributes to its own demise by up-regulating the transcription of S100B as part of a negative feedback loop. This is analogous to what is found for another protein that down-regulates p53, namely hdm2 (human double mutant 2).

Delayed Expression of Apoptosis in X-irradiated Human Leukemic MOLT-4 Cells Transfected with Mutant p53.

The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival.

NEW EMBO MEMBERS' REVIEW: Hsp70 interactions with the p53 tumour suppressor protein

The heat shock proteins (HSPs) are encoded by genes whose expression is substantially increased during stress conditions, such as heat shock, alcohol, inhibitors of energy metabolism, heavy metals, oxidative stress, fever or inflammation. During these conditions, HSPs increase cell survival by protecting and disaggregating stress‐labile proteins (Skowyra et al ., 1990), as well as the proteolysis of the damaged proteins (Wickner et al ., 1999). Under non‐stress conditions, HSPs have multiple housekeeping functions, such as folding and translocating newly synthesized proteins, activation of specific regulatory proteins, including transcription factors, replication proteins and kinases, protein degradation, protein signalling, including steroid hormone activation and tumour immunogenicity, and antigen presentation (for reviews see Helmbrecht et al ., 2000; Jolly and Morimoto, 2000). This broad spectrum of functions gave rise to the term ‘molecular chaperone’, an entity that acts to assist other proteins folding and maturating in the cell. It should also be emphasized that not all HSPs are molecular chaperones and not all chaperones are HSPs (Ellis and Hartl, 1999). HSPs are designated nomenclature according to their approximate molecular weight, e.g. the 70 kDa HSP is known as the molecular chaperone Hsp70. The 70 kDa heat shock‐related proteins comprise a family of highly conserved molecular chaperones that regulate a wide variety of cellular processes during normal and stress conditions (Boorstein et al ., 1994). Hsp70 is one of the most abundant of these proteins, accounting for as much as 1–2% of total cellular protein (Herendeen et al ., 1979). In humans, there are at least 11 distinct genes that code for Hsp70 isoforms, which are located on several different chromosomes (Tavaria et al ., 1996). The major, constitutively expressed hsp70 isoform is called hsc70 (gene product known as the clathrin‐uncoating ATPase or Hsp73) (Welch, 1992). The transcription of inducible forms of hsp70 or hsp72 are under …

The apoptotic and transcriptional transactivation activities of p53 can be dissociated

Previous studies have shown that the apoptotic response of cells following DNA damage requires p53 expression. Wild-type p53 protein levels increase in response to DNA damage and its growth-suppressive action is thought to be mediated by transcriptional activation of the p21/WAF1/CIP1 gene, the product of which is a potent inhibitor of cyclin-dependent kinases. The mechanism by which elevated p53 levels lead to apoptosis is not known, but is believed to involve transcriptional activation of apoptotic genes, such as BAX. We have studied transformed human cells that constitutively express high levels of the R273H mutant p53, which has been reported to lack transcriptional activation activity. We used the inability to induce the p21/Waf1/Cip1 protein as a marker to verify the lack of transcriptional activation activity. Cells expressing the R273H mutant of p53 do not show an increase in p21/Waf1/Cip1 following irradiation with ionizing or UVB radiation. Surprisingly, these cells are very susceptible to induction of apoptosis by UVB radiation, as seen by the formation of a nucleosomal ladder and the proteolytic cleavage of poly(ADP-ribose) polymerase. This suggests that the R273 mutant p53 can function normally in apoptosis but not in transcriptional activation following DNA damage. Furthermore, an inhibitor of RNA polymerase II is a potent inducer of apoptosis in these cells, demonstrating that transcription is not required for apoptosis and suggesting that stalled RNA polymerase II complexes can initiate apoptosis. Interestingly, proteolytic cleavage of p53 occurs during apoptosis in these cells, generating a 45-kDa fragment and liberating the DNA repair helicase binding domain of p53. We propose that the peptide liberated from the carboxy terminus of p53 may contribute to its apoptotic activity, possibly through interaction with the XPB and XPD DNA helicases.

Proteolysis by calpains: a possible contribution to degradation of p53.

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Role of p53 mutation in polyomavirus-induced tumorigenesis.

Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflects the common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.

Expression and sequence analysis of the SDI1/WAF1/CIP1/p21 tumor suppressor gene in prostate cancer cell lines

The SDI1/WAF1/CIP1 gene encodes a M(r) 21,000 protein (p21) that can arrest cell growth by associating with and inhibiting cyclin-dependent kinase complexes necessary for cells to exit G(1). It is a critical downstream effector in the p53 growth control pathway and can be transcriptionally upregulated by increasing levels of wild-type p53 protein. Somatic mutations in the p21 gene have been detected in 17% of primary prostate cancers. In the current study, we examined four prostate cancer cell lines for expression of and mutations in the p21 gene. Transcripts for p21 mRNA were present in all PC cell lines; p21 protein was detected in androgen-dependent LNCaP cells, as well as in androgen-independent DU-145 and TSU-Pr1 cells but not in androgen-independent PC-3 cells. Examination of the entire coding region of the p21 gene by SSCP analysis and direct DNA sequencing did not detect mutations in the coding domains of the p21 gene. These data indicate that mutations of the SDI1/WAF1/CIP1/p21 gene are not present in cultured PC cells and suggest that defects in the p21 gene are infrequent in prostate cancers.

The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis.

The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.

Increased p53 Protein Does Not Correlate to p53 Gene Mutations in Microdissected Human Testicular Germ Cell Tumors

Purpose To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. Materials and Methods We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from paraffin block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. Results In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1 seminoma with a codon 140 silent mutation (no protein alteration). Conclusions Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated.

Increased p53 protein does not correlate to p53 gene mutations in microdissected human testicular germ cell tumors.

To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from paraffin block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1 seminoma with a codon 140 silent mutation (no protein alteration). Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated.

The p53 and mdm-2 genes in human testicular germ-cell tumors.

Mutations in the p53 gene are common in many cancers. They have been documented to occur in about 55% of all cancers of 51 different cell and tissue types. These mutations are accompanied by overexpression of the p53 protein in the nucleus of the cell, and this protein has lost its tumor suppressor function. In this study, 25 testicular germ-cell (TGC) tumors were tested for p53 mutations and the level of p53 protein expression. While 67% of the tumors overproduced the p53 protein in the nucleus of 10-60% of their cells, in all cases the DNA sequence of exons 4-9 of the p53 gene was wild type. In this tumor type, there was apparently no selection pressure for p53 mutations. The mdm-2 gene residues on chromosome 12 (12q13-q14), a chromosome often altered in TGC tumors. mdm-2 gene amplification (2.5- to 10-fold) was detected in three (12%) of these TGC tumors. These three tumors, and eight additional TGC tumors, overexpressed mdm-2 mRNA. There was a good correlation between overexpression of p53 protein and overexpression of mdm-2 mRNA (P = 0.01). This may well result from the fact that the level of mdm-2 mRNA is regulated by the p53 level. These studies demonstrate that TGC tumors fail to be selected for p53 mutations but nonetheless frequently expressed high levels of wild-type p53 protein in the cell nucleus. Perhaps this produces the excellent response to radiation and chemotherapy of these tumors, which generally have a good prognosis. Wild-type p53 may mediate apoptosis in these cells in response to the DNA damage.

The mutational status of p53 protein in gastric and esophageal adenocarcinoma cell lines predicts sensitivity to chemotherapeutic agents

Resistance to chemotherapy remains a serious problem in the successful treatment of gastric and esophageal cancers. DNA-damaging agents alter levels of p53 protein in several cell types and it has been speculated that regulation of p53 can be involved in the resistance or sensitivity of cancer cells to some chemotherapeutic drugs, depending on whether cells have mutant or wild-type p53; however, little is known about the relationship of p53 to drug sensitivity in gastric/esophageal cancers. Here we have examined human gastric/esophageal adenocarcinoma cell lines for p53 mutational status, chemosensitivity to 5-fluorouracil, mitomycin C, and cis-dichlorodiammineplatinum(II), alteration in p53 levels following exposure of cells to these drugs, and the mechanisms involved in regulating p53 levels. Our results indicate that wild-type p53 protein levels increase after treatment with each of these drugs via either post-translational and/or translational mechanisms and that this increase in wild-type p53 appears to be required for effective chemotherapeutic growth control of gastric/esophageal adenocarcinoma cells. In contrast, gastric/esophageal cancer cells expressing either mutated p53 protein or no p53 protein are more resistant to the growth-inhibitory effects of these drugs, despite the fact that drug exposure can also increase mutant p53 levels by a translational mechanism. Thus, these data indicate that the mutational status of p53 is predictive of chemosensitivity of gastric and esophageal adenocarcinomas, and suggest a mechanism in which p53 protein contributes to the cellular response to chemotherapy.