P53 Defect Research Articles

The Prediction of Recurrence in Low-Risk Endometrial Cancer: Is It Time for a Paradigm Shift in Adjuvant Therapy?

Five to 10% of patients with stage IA, grade 1 or 2, endometrioid adenocarcinoma subsequently develop locoregional or distant recurrence. These patients have significantly reduced 5-year survival rates and salvage therapy success rates as low as 40%. The aim of this review is to highlight knowledge gaps that could further refine the risk categories of endometrial carcinoma (EC) and guide future randomized trials of adjuvant therapy for low-risk EC. A systematic search of the literature on PubMed and Medline was conducted using the following search terms: endometrial cancer, endometrial adenocarcinoma, endometrioid adenocarcinoma, low grade, early stage, stage IA, low risk, locoregional recurrence, and relapse. Relevant primary studies were extracted and included in this review. Risk factors for recurrence of low-risk EC were epidemiological (age, body mass index, ethnicity), molecular (DNA MMR, MSI, TP53 mutation and P53 defect, CTNNB1 mutation, PTEN and POLE mutation, L1CAM expression), pathological (positive peritoneal cytology, lymphovascular invasion, tumor size), and others like Ki67-percentage, micro-RNA expression, and hormonal receptor expression. CTNNB1 mutation, L1CAM expression, lymphovascular invasion, and tumor size were identified as significant risk factors for recurrence in low-risk EC. There are subsets of low-risk EC patients at high risk of recurrence and should be suspected when having the following risk factors: positive molecular markers, large tumor size, and lymphovascular invasion. A novel scoring system and randomized controlled trials should be conducted to identify these patients who will benefit most from adjuvant therapy to avoid recurrence.

Abstract A45: Quinacrine induces necrosis of ovarian high-grade serous carcinoma cells by breaking the mitochondria ROS balance

Abstract Ovarian high-grade serous ovarian carcinoma (HGSC) was found to be initiated and promoted by repeated exposure of fimbrial epithelium to ovulatory ROS with resultant intolerable stress of oxidative damages and DNA repair load. Consequential cell apoptosis is overcome on the one hand by quenching excessive ROS by hemoglobin contributed by retrograde menstruation and on the other by upregulation of antioxidant machinery. A dangerous balance is achieved between the oxidative stress and antioxidation and between DNA damage stress and repair. Breaking of this balance on the stress side such as by DNA chelating agent such as cisplatin or inhibitors targeting on DNA repair enzyme such as PARP leads to therapeutic cell killing. In this report, we show ROS enhancing drug such as quinacrine effectively kill HGSC cells and its tubal precursor cells with p53 and Rb defect. Quinacrine is a traditional antimalarial drug with wide antitumor effect in either a single agent or combined with chemotherapy drugs. We show here that quinacrine has a cytotoxic effect in two HGSC cell lines (KURAMOCHI, OVSAHO) and in an HPV E6/E7-immortalized human fimbrial epithelial cells, which are readily fully transformed by ovulatory follicular fluid. Cytotoxicity was observed dose-dependently at concentrations ranging from 2.5μM to 25μM. Flow cytometry analysis indicated a cell necrosis (PI +, annexin V-) 48hr post-treatment. We further found a significant induction of the mitochondrial ROS upon quinacrine treatment, and ROS inhibitor N-acetyl-l-cysteine considerably reversed this cytotoxic effect. The study proves the concept of breaking the balance of mitochondria-ROS is a feasible strategy for the development of a new drug for ovarian HGSC. Note: This abstract was not presented at the conference. Citation Format: Ching-I Huang, Tang-Yuan Chu, Hsuan-Shun Huang. Quinacrine induces necrosis of ovarian high-grade serous carcinoma cells by breaking the mitochondria ROS balance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A45.

Immunohistochemical evaluation of mismatch repair proteins and p53 expression in extrauterine carcinosarcoma/sarcomatoid carcinoma.

IntroductionCarcinosarcoma (CS) is a tumor with components: epithelial (carcinomatous) and mesenchymal (sarcomatous), developing in the mechanism of epithelial-mesenchymal transition. It is known that the p53 defect is a frequent finding in a carcinosarcoma in different anatomical locations, additionally, in a subgroup of uterine CS MMR defect plays a role in the pathogenesis. The aim of this paper was to investigate the frequency of MMR and p53 aberrations in extrauterine CS.Material and methodsTwenty eight extrauterine CS from the lung (n = 8), breast (n = 6), head and neck (n = 5), ovary (n = 3), urinary bladder (n = 3), adrenal gland (n = 1), skin (n = 1), and stomach (n = 1) were stained for hMLH1, PMS2, hMSH2, hMSH6 and p53. The pattern of expression was evaluated separately in carcinomatous and sarcomatous component.ResultsImmunostainings for hMLH1, PMS2, hMSH2 and hMSH6 were positive in all tumors. p53 defect was observed in 19 out of 28 samples (67.85%). In all cases except one (96.42%) there was a concordance between sarcomatoid and carcinomatous components.ConclusionsMMR deficiency does not seem to play a role in the pathogenesis of extrauterine CS. p53 aberrant expression is frequent and almost always consistent in carcinomatous and sarcomatous component.

DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine.

Human lung cancer H1299 (p53-null) cells often display enhanced susceptibility to chemotherapeutics comparing to A549 (p53-wt) cells. However, little is known regarding to the association of DNA damage-response (DDR) pathway heterogeneity with drug sensitivity in these two cells. We investigated the DDR pathway differences between A549 and H1299 cells exposed to 8-chloro-adenosine (8-Cl-Ado), a potential anticancer drug that can induce DNA double-strand breaks (DSBs), and found that the hypersensitivity of H1299 cells to 8-Cl-Ado is associated with its DSB overaccumulation. The major causes of excessive DSBs in H1299 cells are as follows: First, defect of p53-p21 signal and phosphorylation of SMC1 increase S phase cells, where replication of DNA containing single-strand DNA break (SSB) produces more DSBs in H1299 cells. Second, p53 defect and no available induction of DNA repair protein p53R2 impair DNA repair activity in H1299 cells more severely than A549 cells. Third, cleavage of PARP-1 inhibits topoisomerase I and/or topoisomerase I-like activity of PARP-1, aggravates DNA DSBs and DNA repair mechanism impairment in H1299 cells. Together, DDR pathway heterogeneity of cancer cells is linked to cancer susceptibility to DNA damage-based chemotherapeutics, which may provide aid in design of chemotherapy strategy to improve treatment outcomes.

Analysis of prognostic significance of merkel cell polyomavirus in chronic lymphocytic leukemia.

Merkel cell polyomavirus (MCPyV), a ubiquitous DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukemia (CLL). Previous studies have reported conflicting results on the frequency and potential pathogenetic role of MCPyV in CLL. The aim of this study was to evaluate MCPyV's association with CLL and its prognostic significance. Between 2006 and 2013, DNA samples obtained from CLL patients (n= 119) before treatment were tested for MCPyV using quantitative real-time polymerase chain reaction analysis and verified by gel electrophoresis. Only samples testing positive by both methods were considered valid. We found that 13 (11%) of 119 CLL cases were positive for MCPyV. Between the groups of MCPyV-positive and -negative patients, there was no significant difference in the sex, age, cytogenetics, presence of p53 defect, or immunoglobulin heavy chain (IGHV) mutational status. In the subset of MCPyV-negative patients, advanced Rai stage (III to IV) was found more frequently, and therapy was initiated more often. There was no difference in overall response rate, median progression-free survival, and overall survival between both groups. We did not observe any new positivity after treatment in initially MCPyV-negative patients. This study provides the first analysis of the prognostic role of MCPyV in CLL. MCPyV occurrence seems to be a relatively rare event during the course of CLL. MCPyV is also unlikely to influence the outcome of CLL patients.

Lack of functional p53 renders DENSpm-induced autophagy and apoptosis in time dependent manner in colon cancer cells.

Polyamines (PAs), such as putrescine, spermidine and spermine, are alkyl-amines that are essential for cell growth, proliferation, differentiation and cancer progression in eukaryotic cells. A designed PA analogue; DENSpm, induces cell cycle arrest, inhibits proliferation and induces apoptosis in melanoma, breast, prostate, lung and colon cancer cells. Although the mechanism by which DENSpm induces apoptosis has been examined, the effect of DENSpm on autophagy has not been investigated yet. Therefore, in this study, our objective was to determine the role of p53 in the DENSpm-induced autophagy/apoptotic regulation in a time-dependent manner in colon cancer cells. Exposure of HCT 116 colon cancer cells to DENSpm decreased cell viability in a dose- and time-dependent manner. However, the p53 mutant, SW480, and deficient HCT 116 p53(-/-) cells were more resistant to DENSpm treatment compared to HCT 116 p53(+/+) cells. The resistant profile caused by p53 defect also caused a cell type-specific response to PA pool depletion and SSAT overexpression. In addition to PA depletion, DENSpm induced apoptosis by activating the mitochondria-mediated pathway in a caspase-dependent manner regardless of p53 expression in colon cancer cells. Concomitantly, we determined that DENSpm also affected autophagy in HCT 116 p53(+/+), SW480 and HCT 116 p53(-/-) colon cancer cells for different periods of exposure to DENSpm. Therefore, this study revealed that effect of DENSpm on cell death differs due to p53 protein expression profile. In addition, DENSpm-induced autophagy may be critical in drug resistance in colon cancer cells.

Microrna Expression in Fludarabine-Refractory CLL Implicates Independent Mechanisms of Resistance and Is Associated with Response and Progression Free Survival After Alemtuzumab Treatment: Results From the CLL2H Trial.

Abstract 2874Although novel treatment options have improved response rates and survival in CLL, the portion of cases with refractory disease remains considerably high and management is challenging. The humanized anti-CD52 monoclonal antibody alemtuzumab is standard treatment in refractory CLL and shows efficacy irrespective of the underlying genomic aberrations or mutation status of TP53. However, only about half of all refractory cases can be explained by TP53 mutation/deletion and treatment remains unsatisfactory.To elucidate pathogenic and prognostic factors, we performed microRNA expression analyses on a set (n=42) of fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3×30 mg alemtuzumab s.c. per week, max 12 weeks). Profiling was done with the Agilent microRNA microarray platform based on the Sanger miRBase Release 14.0. Statistical analysis was performed with the R software version 2.15.1. Normalization of the arrays was conducted by using the least-variant set (LVS) of genes method and by quantile normalization.When filtering the expression data based on standard deviation (sd>0.5), we found 131 microRNAs showing greatest variability in expression levels across all arrays. Subsequent unsupervised bootstrap clustering (mean centered; distance: pearson correlation; agglomeration rule: average linkage) resulted in two stable major clusters. Cluster 1 contained more cases with p53 defect which were observed in 56% (TP53 mutation 7/16, 17p deletion 6/16) vs. 27% (TP53 mutation 6/26, 17p deletion 5/26). Cluster 2 was slightly enriched for IGHV unmutated cases in 61% (16/26) vs. 50% (8/16). Median levels of thymidine kinase were 31.6 U/L in cluster 1 and 39.6 U/L in cluster 2, beta2-microglobulin levels were similar in both clusters with a median of 4.3 and 4.4 mg/L. Expression of miR-34a, a microRNA known to execute p53-dependent cell cycle arrest, as well as induction of senescence and apoptosis, was predominantly low in cluster 1. Cases with low miR-34a expression in cluster 1 and without altered p53 (no 17p deletion and/or TP53 mutation) showed a selective upregulation of microRNAs known to be involved in proliferation, neoangiogenesis and inhibition of apoptosis by targeting genes like CDKN1A, E2F, BIM and PTEN. Interestingly, these microRNAs have been reported to be repressed by p53 under physiologic conditions, pointing to alternative mechanisms of p53 pathway inactivation. With regard to treatment response, cluster 1 was significantly enriched for better response (partial response (PR) n=9 (56.25%); stable disease (SD) n=5 (31.25%); progressive disease (PD) n=2 (12.5%)). In contrast cluster 2 (n=26) was dominated by cases with inferior response (PR n=4 (15%); SD n=13 (50%); PD n=9 (35%)). Progression free survival (PFS) was significantly longer in cluster 1 (median 331 days vs. 145 days, p=0.0171, Figure 1). Overall survival was in trend longer in cluster 1 (median 760 days vs. 513 days, p=0.2480).In conclusion, microRNA expression in fludarabine-refractory CLL shows high variability likely induced by independent mechanisms of resistance. Altered microRNA expression might mimic effects similar to TP53 mutation or 17p deletion even in situations when p53 itself is not functionally disrupted. Of note, unsupervised clustering identified two major clusters that showed a surprising prognostic relevance in fludarabine-refractory CLL treated with alemtuzumab. [Display omitted] Disclosures:Stilgenbauer:Genzyme: Honoraria, Research Funding.

Down-Regulation of AP-4 Inhibits Proliferation, Induces Cell Cycle Arrest and Promotes Apoptosis in Human Gastric Cancer Cells

BackgroundAP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells.MethodsTwo specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level.ResultsThe expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-xL was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III.ConclusionsThese results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer.

A p53 Defect Sensitizes Various Stages of B Cell Development to Lymphomagenesis in Mice Carrying an IgH 3′ Regulatory Region-Driven c-myc Transgene

Although c-myc is classically described as the driving oncogene in Burkitt's lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3'RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10-12 mo). To reproduce c-myc-induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3'RR with p53(+/-) mice. Double transgenic c-myc-3'RR/p53(+/-) mice developed lymphoma with short latency (2-4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3'RR/p53(+/-) mice was widened, including nonactivated (CD43(-)) BL, activated (CD43(+)) BL, MCL-like lymphoma, and PCL, thus showing that 3'RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53(+/-) mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc-induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.

UVB-Induced p21 degradation promotes apoptosis of human keratinocytes

Skin cancer is the most common cancer in the United States. Ultraviolet B (UVB) radiation in sunlight is the major environmental factor causing skin cancer. p21, a p53-inducible protein, plays an important role in cell cycle, DNA repair, and apoptosis. Here we have investigated the effect of UVB radiation on p21 and its molecular mechanisms and function in human HaCaT keratinocytes, which we used as a premalignant cellular model because normal skin harbors numerous clones of p53-mutated keratinocytes. We found that in human HaCaT keratinocytes UVB induces rapid p21 down-regulation via a proteasomal degradation mechanism. In p53-defective HaCaT cells, the p21 protein levels remain decreased at a later time post-UVB, but in normal human and mouse epidermal keratinocytes with wild-type p53 the p21 levels are initially reduced but later increase post-UVB. These findings indicate that loss of p53 function leads to sustained p21 down-regulation in response to UVB damage. Degradation of p21 following UVB radiation does not require ATR, ATM, or both, because either the ATR/ATM inhibitor caffeine or siRNA knockdown of ATR, ATM, or both failed to reverse p21 degradation. However, inhibiting MDM2 or GSK3β partially reduced UVB-induced p21 degradation, while inhibiting both enzymes completely prevented it. Restoring the p21 protein levels in UVB-irradiated keratinocytes reduced apoptosis. Although at the molecular level increasing p21 expression has no effect on the protein levels of the Bcl-2 family members, it enhances the activation of AKT, a critical survival pathway to protect cells from apoptosis. Our results suggest a distinct mechanism of p21 degradation in keratinocytes by UVB, and this p21 degradation may significantly enhance UVB-induced apoptosis of premalignant keratinocytes with a p53 defect to eliminate damaged cells and therefore prevent skin cancer development.

DNA double-strand break repair activities in mammary epithelial cells--influence of endogenous p53 variants

Intriguingly, all 10 breast cancer susceptibility genes known today are directly or indirectly related to DNA double-strand break (DSB) repair suggesting a critical role of DSB repair dysfunction in the etiology of this tumor entity. We and others had previously provided evidence indicating that the breast cancer susceptibility gene product p53 controls DSB repair. Experiments with ectopically expressed proteins showed that oncogenic mutants of p53 deregulate homologous recombination (HR) and possibly also non-homologous end joining (NHEJ). Here, we systematically analyzed the role of different p53 variants endogenously expressed in a series of mammary epithelial cell lines. We provide evidence that endogenous wild-type p53 represses HR, particularly between short homologies that strengthens the idea of a quality control mechanism underlying HR regulation. To a lesser extent, p53 also downregulates microhomology-mediated NHEJ and single-strand annealing. Our data also suggest that repression of NHEJ regulation may require the extreme C-terminus, whereas the oligomerization and core domains are involved in HR regulation. We show that depending on the individual mutation, p53 mutants retain more or less partial DSB repair downregulatory activities when compared with loss of p53. All in all, relative effects on distinct DSB repair pathways and discrimination between HR substrates with perfectly versus imperfectly homologous sequences represent good markers for a p53 defect due to a specific mutation. Thus, advanced DSB repair analysis may serve as a novel assay for the functional classification of p53 mutations.

Abrogation of the Transactivation Activity of p53 by BCCIP Down-regulation

The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in approximately 50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-interacting protein, is required for the transactivation activity of wild type p53. In p53 wild type cells, BCCIP knock down by RNA interference diminishes the transactivation activity of p53 without reducing the p53 protein level, inhibits the binding of p53 to the promoters of p53 target genes p21 and HDM2, and reduces the tetrameric formation of p53. These data demonstrate a critical role of BCCIP in maintaining the transactivation activity of wild type p53 and further suggest down-regulation of BCCIP as a novel mechanism to impair the p53 function in cells harboring wild type p53.

P53-based cancer therapies: is defective p53 the Achilles heel of the tumor?

The tumor suppressor protein p53 plays a pivotal role in the DNA damage response and is defective in >50% of human tumors, which has generated substantial interest in developing p53-targeted cancer therapies. Various therapeutic rationales targeting p53 are currently under investigation including attempts to both activate and inhibit p53. Elevation of p53 can be achieved by either reintroducing an exogenous p53 gene or by blocking its association with its negative regulator hDM2. An alternate approach involves reverting mutant p53 to its wild-type conformation. Inhibition of p53 activity can be achieved either by preventing p53-mediated gene expression or by inhibiting the mitochondrial pro-apoptotic interactions of p53. These approaches are based on the concept that activation of p53 in a tumor is cytotoxic while inhibition of p53 in normal cells will protect the patient. However, activation of p53 also induces cell cycle arrest that can protect most normal cells from DNA damage, and this is the reason why many p53-defective tumors are more sensitive to DNA damage. The development of cell cycle checkpoint inhibitors to abrogate DNA damage-induced arrest builds on this observation as p53-defective cells appear particularly sensitive. Thus, normal cells are protected from premature entry into mitosis and the subsequent mitotic catastrophe induced by checkpoint inhibitors, while p53-defective tumor cells are destroyed. These contradictory approaches must be resolved if we are to take full advantage of the frequent p53 defect in tumors.

Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation. The carbon ions were accelerated to 135 MeV/u by the RIKEN Ring Cyclotron. The p53 defect markedly enhanced the Spi(-) mutant frequency (MF) in the kidneys of mice exposed to C-ion irradiation: the Spi(-) MF increased 4.4- and 2.8-fold over the background level after irradiation in p53(-/-) and p53(+/+) mice, respectively. There was no significant difference in the background Spi(-) MF between p53(-/-) and p53(+/+) mice. Sequence analysis of the Spi(-) mutants indicated that the enhancement of kidney Spi(-) MF in p53(-/-) mice was primarily due to an increase in complex or rearranged-type deletions. In contrast to the kidney, the p53 defect had no effect on the Spi(-) MF in liver: Spi(-) MF increased 3.0- and 2.7-fold after the irradiation in p53(-/-) and p53(+/+) mice, respectively. Our results suggest that p53 suppresses deletion mutations induced by heavy-ion irradiation in an organ-specific manner.

Spontaneous development of drug resistance: mismatch repair and p53 defects in resistance to cisplatin in human tumor cells.

The contributions of defective mismatch repair and mutated p53 to cisplatin resistance of human tumor cells were analysed. Mismatch repair defects were not associated with a predictable degree of resistance among several tumor cell lines. Repair defective variants of the A2780 ovarian carcinoma cell line which were isolated by selection for a methylation tolerant phenotype and did not express the hMLH1 mismatch repair protein, were highly resistant to cisplatin. Their cisplatin resistance was not a simple consequence of the mismatch repair defect. They were members of a drug-naive subpopulation of A2780 in which a silent hMLH1 gene accompanies a mutated p53. Two complementary approaches indicated that each defect contributes to cisplatin resistance independently and to a different extent. Firstly, separate introduction of a p53 defect into A2780 cells significantly increased their cisplatin resistance; defective hMLH1 provided less extensive protection. Secondly, azadeoxycytidine reactivation of the silent hMLH1 gene or expression of a transfected hMLH1 cDNA sensitized the doubly hMLH1/p53 deficient cells only slightly to cisplatin. Both approaches indicate that defective p53 status is a major determinant of cisplatin resistance and defective mismatch repair is a minor, and independent, contributor. The data have implications for the development of intrinsic cisplatin resistance.

Enhancement of urinary bladder carcinogenesis in nullizygous p53-deficient mice by N-butyl- N-(4-hydroxybutyl)nitrosamine

We recently reported p53 mutations to be frequent in mouse invasive urinary bladder carcinomas, with and without metastasis. However, the role of p53 dysfunctions during carcinogenesis remains unclear. In the present study, heterozygous and nullizygous p53-deficient mice and their littermates were treated with the urinary bladder carcinogen, N-butyl- N-(4-hydroxybutyl)-nitrosamine (BBN), at a concentration of 0.01% in the drinking water throughout the experiment. This markedly accelerated urinary bladder carcinogenesis but not development of other tumors in the nullizygous p53-deficient mice. Thus the appearance of neoplastic urothelial lesions in nullizygotes (at day 60 of the experiment) was earlier than in wild-type mice and heterozygotes (at day 125). Moreover, malignant vascular tumors (hemangiosarcomas (HS)) were found in all four nullizygotes killed later than day 108. Mutational inactivation of the wild-type allele was not apparent in either the single transitional cell carcinoma observed in a wild-type mouse and a hemangiosarcoma in a heterozygote. Overall, it can be concluded that the number of normal p53 alleles is a significant determining factor in the susceptibility of urothelial cells to carcinogens. The role of the p53 defect in mouse urinary bladder carcinogenesis may thus be to diminish the threshold for occurrence of additional genetic alterations.

Human Papillomavirus-Induced Carcinogenesis with p53 Deficiency in Mouse: Novel Lymphomagenesis in HPV16E6E7 Transgenic Mice Mimicking p53 Defect

To investigate the transforming activity of human papillomavirus (HPV) E6 and E7 genesin vivo, we previously established transgenic mouse lines containing HPV16E6E7, in which male mice develop a Leydig cell tumors with a very high incidence. Because HPV-induced carcinogenesis is highly related to p53, we changed the dose of p53 gene in the transgenic lines by the mice crossing with p53-disrupted mice. The transgenic mice with homozygous wild-type p53 alleles developed only the testicular tumor, whereas novel T cell lymphomagenesis occurred in the heterozygous p53–disrupted E6E7 (p53+/−E6E7) transgenic mice. In this tumor and even in the normal spleen, the absence of p53 protein was observed, whereas the p53 mRNA was expressed with a normal size, suggesting the degradation of p53 protein in these tissues. These results suggest that HPV16E6 could stimulate p53 protein degradation in mouse cells and induced the lymphomagenesis in a manner indistinguishable from p53 deficiency.

Alteration in p53 pathway and defect in apoptosis contribute independently to cisplatin-resistance.

The accumulation of molecular genetic defects selected during the adaptation process in the development of cisplatin-resistance was studied using progressive cisplatin-resistant variants (L1210/DDP2, L1210/DDP5, L1210/DDP10) derived from a murine leukemia cell line (L1210/0). Of these cell lines, only the most resistant L1210/DDP10 was cross-resistant to etoposide and deficient in apoptosis induced by these two drugs, indicating that resistance to DNA-damaging agents correlates with a defect in apoptosis. This defect was tightly associated with the loss of a Ca2+/Mg2+-dependent nuclear endonuclease activity present in the less cisplatin-resistant cells. Evidence is presented that p53-dependent function (a) is lost not only in the apoptosis defective L1210/DDP10 cells, but also in the apoptosis susceptible L1210/DDP5 cells; (b) is unrelated to drug-induced cell cycle perturbations. These results suggest that deficiency in the p53 pathway and resistance to DNA-damaging agents due to a defect in apoptosis are independent events.