Absence Of Wild-type P53 Research Articles

Mutant p53 elicits context-dependent pro-tumorigenic phenotypes

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular “hot spots” in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

MDM2, MDMX, and p73 regulate cell-cycle progression in the absence of wild-type p53

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2-Murine Double Minute X (MDM2-MDMX) heterodimer. The role of MDM2-MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

Abstract A55: Overexpression of the basic helix-loop-helix transcription factor, E47, promotes p16-independent senescence in established and patient-derived xenograft lines

Abstract In normal pancreatic acinar cells, acquisition of activating mutations in Kras leads to protective oncogene-induced-senescence (OIS) (Collado et al, Nature 2005). This barrier to malignant transformation is lost upon subsequent mutation and LOH of p53, DNA-methylation based silencing of the p16 locus, and pancreatitis (Guerra et al, Cancer Cell 2011). We have previously shown that pancreatic ductal adenocarcinoma (PDA) tumors express high levels of ID3, an inhibitor of bHLH transcription factor activity. Moreover, overexpression of ID3 alone in human exocrine cells was sufficient to induce cell cycle entry suggesting that the bHLH/ID3 axis is a powerful arbiter of cell cycle status in vitro and in vivo (Lee et al, Molecular Cancer Research 2011). Here we report that restoring bHLH activity in aggressive PDA cells reinstates senescence in the absence of wild-type p53 or p16 in both established PDA lines and patient-derived xenograft lines. Previously we showed that overexpression of the bHLH protein, E47, in established cell lines - Panc-1, Bx-PC3 and MiaPaCa-2 - induced growth arrest through a mechanism that involved upregulation of p21 expression (Kim et al, Pancreas 2015). Here we report that two patient-derived pancreatic cancer cell lines - AA0779E from a primary human PDA, and AA1334E, a liver metastasis from a primary human PDA, also stopped proliferating within 48 hours in response to high levels of E47. We used RNA-sequencing to characterize E47 induced genome-wide changes in the transcriptomes of all five cell lines and found that the most significant and consistent alterations occurred in expression of a large cohort of genes that code for regulators of the cell cycle. In all lines, p21 was among the most highly upregulated. In addition, the RNA-sequencing data revealed that E47 restored normal expression of genes that are specifically linked to senescence, including laminB1, Dec1/bHLHe40 and CENP-A. Moreover, all of the cell lines underwent extensive senescence-associated changes in cell size and morphology concomitant with induction of senescence-associated beta-galactosidase activity (SA-beta-gal). In conclusion, we have shown that overexpressed E47 initiates a senescence program that is independent of wild-type p53 or p16 in all PDA cell lines examined including those derived from a liver metastasis of PDA. Interestingly, Panc-1 and AA0779E cells remained stably arrested for weeks in culture while Bx-PC3, MiaPaCa-2, and AA1334E cells died within 96 hours. This outcome is consistent with a model in which genetic variability among lines determines whether the cells fully execute the cellular senescence program or succumb to cell death after initiation. Importantly, since most patients present with disseminated disease, the data suggest that a therapy aimed at restoring bHLH activity could be effective in halting growth of both primary tumors and metastases. Citation Format: Kathleen M. Scully, Reyhaneh Lahmy, Heejung Kim, Andrew Lowy, Pamela Itkin-Ansari.{Authors}. Overexpression of the basic helix-loop-helix transcription factor, E47, promotes p16-independent senescence in established and patient-derived xenograft lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A55.

Splice variants of MDM2 in oncogenesis.

Many types of human cancers overexpress MDM2 protein. A common characteristic among these cancers is an associated increase in mdm2 splice variants. Provided here is a comprehensive list, based on a literature review, of over 70 mdm2 variants. These variants are grouped according to in-frame versus out-of-frame status and their potential (or ability) to be translated into isoform proteins. We describe the putative functions for these mdm2 splice variant mRNAs, as well as the mechanistic drivers associated with increased mdm2 transcription and splicing. The paradoxical signal transduction functions of the most commonly studied variants mdm2-a,-b and -c are addressed for their outcomes in the presence and absence of wild-type p53. These outcomes vary from tumor promotion to growth arrest. Finally, we present issues in the detection of endogenous MDM2 protein and how many of the antibodies commonly used to detect MDM2 do not present a full picture of the cellular representation of the isoform proteins. This review provides a focusing lens for individuals interested in learning about the complexities of mdm2 mRNAs and their protein isoforms as well as the roles MDM2 isoforms may play in cancer progression.

Not all p53 gain-of-function mutants are created equal

Not all p53 gain-of-function mutants are created equal

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug.

Abstract 2987: Oncogenic Wip1 is activated and negatively regulates DNA-damage induced apoptosis in p53-null cancer cells

Abstract Wip1 (wild-type p53-induced phosphatase, or PPM1D) belongs to the protein phosphatase type 2C family and its expression is induced by p53 in response to various stresses. Wip1 is known to dephosphorylate cellular target proteins such as p38 MAPK, p53, and uracil DNA glycosylase that are involved in DNA-repair and cell-cycle-checkpoint pathways. Here, we show that after methylmethane sulfonate (MMS) treatment Wip1 is induced in various breast-cancer cell lines in the absence of wild-type p53 and p38 MAPK activity. Wip1 expression in response to MMS was not blocked by the p38 MAPK inhibitor SB202190 or in p38-silenced MCF-7 cells using p38 MAPK small-interfering RNA (siRNA). The Wip1 promoter displayed great induction in breast cancer cell lines, which have DNA-binding deficient p53 mutants, and p53-null cancer cells such as Saos-2, A549-E6 and HCT116(p53−/−), whereas a control promoter, p53-Luc, showed the least promoter activity after MMS treatment. The mutation of p53 response element in the Wip1 promoter decreased its activity in both unstressed and stressed cells, but the overall level of fold-induction was similar. In addition, cell death was greatly increased in Wip1-siRNA and MMS treated cells in comparison with scrambled siRNA and MMS treated cells in the both cell lines of HCT116 (p53 +/+) and HCT116 (p53−/−). Our data indicate that Wip1 induction in response to various stimuli is not solely controlled by p53, and other damage response signaling appears to compensate for p53. Furthermore, Wip1 negatively regulates DNA-damage induced cell death regardless of the presence wild-type p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2987.

Drug discovery targeting epigenetic codes: The great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis

UHRF1 plays a central role in transferring methylation status from mother cells to daughter cells. Its SRA domain recognizes hemi-methylated DNA that appears in daughter DNA strands during duplication of DNA. UHRF1 recruits DNMT1 to the site and methylates both strands. UHRF1 also binds to HDAC1 and di- and tri-methyl K9 histone H3, ubiquitinates histone H3, and associates with heterochromatin formation, indicating that UHRF1 links histone modifications, DNA methylation, and chromatin structure. UHRF1 is a direct target of E2F1 and promotes G1/S transition. The tumor suppressor p53, which is deficient in 50% of cancers, down-regulates UHRF1 through up-regulation of p21/WAF1 and subsequent deactivation of E2F1. The expression levels of UHRF1 are up-regulated in many cancers, probably partially because of the absence of wild type p53, but it is probably regulated by several other factors. Knockdown of UHRF1 expression in cancer cells suppressed cell growth, suggesting that UHRF1 can be a useful anticancer drug target. Recently, it was revealed that UHRF1 plays important roles not only in carcinogenesis, but also in toxoplasmosis, which is occasionally fatal to people with a weakened immune system, and can cause blindness in the major pathology of ocular toxoplasmosis. Toxoplasma gondii, which causes toxoplasmosis, utilizes UHRF1 to control the cell cycle phase and enhance its proliferation. Thus, knockdown of UHRF1 can be effective at stopping the proliferation of the parasites in infected cells. In this review, we discuss several possible methods that can inhibit the multiple unique functions of UHRF1, which can be utilized for treating cancers and toxoplasmosis.

MDM2 Acts Downstream of p53 as an E3 Ligase to Promote FOXO Ubiquitination and Degradation

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.

HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function

Nutlin-3, a small molecule inhibitor, activates p53 by disrupting p53-HDM2 association. In this study, we found that Nutlin-3 suppressed cell growth and induced apoptosis in the absence of wild-type p53, suggesting a p53-independent mechanism for Nutlin-3-induced cell death. Like p53, its homolog p73 transactivates proapoptotic genes and induces cell death. Since HDM2, a key negative regulator of p53, also binds to and inhibits p73, we asked whether p73 could mediate Nutlin-3-induced apoptosis. We demonstrate that Nutlin-3 inhibits endogenous binding between the proapoptotic p73 isoform TAp73alpha and HDM2 in p53-null cells. Dissociation of p73 and HDM2 leads to increased p73 transcriptional activity with upregulation of p73 target genes noxa, puma and p21, as well as enhanced apoptosis. p73 knockdown by siRNA results in rescue of Nutlin-3-treated cells, indicating that Nutlin-3-induced apoptosis is, at least in part, p73 dependent. In addition, Nutlin-3 treatment increases TAp73alpha protein levels with prolongation of p73 half-life. These results provide the first evidence that Nutlin-3 disrupts endogenous p73-HDM2 interaction and enhances the stability and proapoptotic activities of p73 and thus, provides a rationale for the use of Nutlin-3 in the large number of human tumors in which p53 is inactivated.

Extreme sensitivity to Yondelis® (Trabectedin, ET‐743) in low passaged sarcoma cell lines correlates with mutated p53

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.

Radiation-Induced Caspase-8 Mediates p53-Independent Apoptosis in Glioma Cells

Malignant gliomas are almost uniformly fatal and display exquisite radiation resistance. Glioma cells lacking wild-type (WT) p53 function are more susceptible to radiation-induced apoptosis than their isogenic counterparts expressing WT p53. We explored the mechanisms of such apoptosis and found that, in the absence of WT p53, radiation increases caspase-8 expression and activity. Inhibition of caspase-8 expression using caspase-8 antisense or small interfering RNA (siRNA) oligonucleotides partially blocks radiation-induced apoptosis. In contrast, inhibition of the mitochondrial death pathway by expression of Bcl-2 has no effect on radiation-induced caspase-8 activity or apoptosis. Our data indicate that, in contrast to commonly accepted models of p53-dependent radiation-induced apoptosis, in our cell system, radiation relies on caspase-8 activity to help mediate p53-independent cell death. In a system of inducible E2F1 activity, E2F1 activated caspase-8 and, accordingly, decreased cellular viability, effects that were abolished by caspase-8 siRNA. In this model, in the absence of WT p53, p21Cip1 is not induced, and E2F1 activity is sustained and allows transcription and activation of caspase-8. This model may explain why p53 mutations in adult gliomas paradoxically correlate with improved survival and enhanced response to radiation.

Identification of an Aberrantly Spliced Form of HDMX in Human Tumors: A New Mechanism for HDM2 Stabilization

The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.

Aminothiol WR1065 induces differential gene expression in the presence of wild-type p53

The aminothiol WR1065 exerts selective cytoprotective effects in normal cells compared to cancer cells and has clinical applications for the protection of normal cells in cancer patients undergoing radio- or chemotherapy. There is evidence that p53 is activated in response to WR1065. To examine the effects of WR1065 on the signalling pathways controlled by p53, isogeneic human colon carcinoma cell lines (HCT116) differing only in the presence or absence of wild-type p53 were used. Treatment with WR1065 resulted in G1 cell cycle arrest in the p53-positive cell line but not in the p53-negative cell line. Long-term exposure resulted in minimal apoptosis of either cell line. Changes in gene expression in p53-positive or -negative cells treated with WR1065 were examined using commercial human stress and cancer gene arrays (Clontech Atlas arrays). Genes found to be specifically upregulated in a p53-dependent manner included coproporphyrinogen oxidase, ICErel-II cysteine protease, macrophage inhibitory cytokine-1 (also known as placental transforming growth factor beta), S100A4, and Waf1/p21. However, most proapoptotic genes typically upregulated by p53 in response to DNA damage were not activated. These studies show that WR1065 specifically modulates a subset of p53 target genes in a colon carcinoma cell line, consistent with the observation that this agent elicits essentially p53-dependent, cell cycle arrest responses.

KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells

A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.

Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan.

To elucidate mechanisms of resistance to chemotherapies currently used in the first-line treatment of advanced colorectal cancer, we have developed a panel of HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic colorectal cancer cell lines resistant to the antimetabolite 5-fluorouracil (5-FU), topoisomerase I inhibitor irinotecan (CPT-11), and DNA-damaging agent oxaliplatin. These cell lines were generated by repeated exposure to stepwise increasing concentrations of each drug over a period of several months. We have demonstrated a significant decrease in sensitivity to 5-FU, CPT-11, and oxaliplatin in each respective resistant cell line relative to the parental line as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, with increases in IC(50 (72 h)) concentrations ranging from 3- to 65-fold. Using flow cytometry, we have also demonstrated compromised apoptosis and cell cycle arrest in 5-FU-, oxaliplatin-, and CPT-11-resistant cell lines compared with the parental lines after exposure to each drug. In addition, we found that resistance to 5-FU and oxaliplatin was higher in parental p53(-/-) cells compared with parental p53(+/+) cells, with an approximately 5-fold increase in IC(50 (72 h)) for each drug. In contrast, the IC(50 (72 h)) doses for CPT-11 were identical in the p53 wild-type and null cell lines. Furthermore, apoptosis after treatment with 5-FU and oxaliplatin, but not CPT-11, was significantly reduced in parental p53(-/-) cells compared with parental p53(+/+) cells. These data suggest that p53 may be an important determinant of sensitivity to 5-FU and oxaliplatin but not CPT-11. Using semiquantitative reverse transcription-PCR, we have demonstrated down-regulation of thymidine phosphorylase mRNA in both p53(+/+) and p53(-/-) 5-FU-resistant cells, suggesting that decreased production of 5-FU active metabolites may be an important resistance mechanism in these lines. In oxaliplatin-resistant cells, we noted increased mRNA levels of the nucleotide excision repair gene ERCC1 and ATP-binding cassette transporter breast cancer resistance protein. In CPT-11-resistant cells, we found reduced mRNA levels of carboxylesterase, the enzyme responsible for converting CPT-11 to its active metabolite SN-38, and topoisomerase I, the SN-38 target enzyme. In addition, we noted overexpression of breast cancer resistance protein in the CPT-11-resistant lines. These cell lines are ideal tools with which to identify novel determinants of drug resistance in both the presence and absence of wild-type p53.

Transcriptional Activation of the MUC2 Gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.

Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo R marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53 +/− mouse fibroblasts show elevated levels of homologous recombination compared to their p53 +/+ counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

The progressing clinical utility of DNA microarrays.

The progressing clinical utility of DNA microarrays.

Differential gene expression in mouse mammary adenocarcinomas in the presence and absence of wild type p53.

The tumor suppressor p53 transcriptionally regulates a large number of target genes that may affect cell growth and cell death pathways. To better understand the role of p53 loss in tumorigenesis, we have developed a mouse mammary cancer model, the Wnt-1 TG/p53 model. Wnt-1 transgenic females that are p53-/- develop mammary adenocarcinomas that arise sooner, grow faster, appear more anaplastic, and have higher levels of chromosomal instability than their Wnt-1 transgenic p53+/+ counterparts. In this study, we used several assays to determine whether the presence or absence of p53 affects gene expression patterns in the mammary adenocarcinomas. Most of the differentially expressed genes are increased in p53+/+ tumors and many of these represent known target genes of p53 (p21WAF/C1P1, cyclin G1, alpha smooth muscle actin, and cytokeratin 19). Some of these genes (cytokeratin 19, alpha smooth muscle actin, and kappa casein) represent mammary gland differentiation markers which may contribute to the inhibited tumor progression and are consistent with the more differentiated histopathology observed in the p53+/+ tumors. Several differentially expressed genes are growth regulatory in function (p21, c-kit, and cyclin B1) and their altered expression levels correlate well with the differing growth properties of the p53+/+ and p53-/- tumors. Thus, while tumors can arise and progress in the presence of functioning wild type p53, p53 may directly or indirectly regulate expression of an array of genes that facilitate differentiation and inhibit proliferation, contributing to a more differentiated, slow growing, and genomically stable phenotype.