Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/ß-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3'-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

Progestin and estrogen potency of combination oral contraceptives and endometrial cancer risk.

OBJECTIVE: Using data from a case-control study of endometrial cancer, we investigated the relationship between the progestin and estrogen potency in combination oral contraceptives (OCs) and the risk of developing endometrial cancer. METHODS: Subjects included 434 endometrial cancer cases and 2,557 controls identified from the Cancer and Steroid Hormone (CASH) study. OCs were classified into four categories according to the individual potencies of each hormonal constituent (high versus low estrogen or progestin potency). Logistic regression was used to evaluate associations between endometrial cancer risk and combination OC formulations. RESULTS: With non-users as the referent group, use of OCs with either high potency progestin [odds ratio for endometrial cancer (OR)=0.21, 95% confidence interval (CI)=0.10 to 0.43] or with low potency progestin (OR=0.39, 95% CI=0.25 to 0.60) were both associated with a decreased risk of endometrial cancer. Overall high progestin potency OCs did not confer significantly more protection than low progestin potency OCs (OR=0.52, 95% CI=0.24 to 1.14). However, among women with a body mass index of 22.1 kg/m2 or higher, those who used high progestin potency oral contraceptives had a lower risk of endometrial cancer than those who used low progestin potency oral contraceptives (OR=0.31, 95% CI=0.11 to 0.92) while those with a BMI below 22.1 kg/m2 did not (OR=1.36, 95% CI=0.39 to 4.70). CONCLUSION: The potency of the progestin in most OCs appears adequate to provide a protective effect against endometrial cancer. Higher progestin-potency OCs may be more protective than lower progestin potency OCs among women with a larger body habitus.

Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression.

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

Identification of epidermal growth factor receptor- Grb2-associated binder-1-SHP-2 complex formation and its functional loss during neoplastic cell progression.

The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.

Mutational analysis of the PTEN gene in human uterine sarcomas.

OBJECTIVE: Uterine sarcomas are rare, lethal cancers, and little is known about their molecular etiology. The PTEN gene is located on chromosome 10q23.3, a region that displays frequent loss of heterozygosity in human uterine sarcomas. PTEN mutations have been described in 40% to 60% of uterine adenocarcinomas. To determine whether the PTEN gene is involved in the pathogenesis of uterine sarcoma, we analyzed deoxyribonucleic acid from uterine sarcomas and cell lines. STUDY DESIGN: Single-strand conformation analysis and direct sequencing of deoxyribonucleic acid were used to screen for PTEN mutations. RESULTS: Silent polymorphisms were detected in 2 of 36 primary uterine sarcomas. A 4-base pair deletion and a point mutation producing a stop codon were identified in 1 cell line. CONCLUSIONS: Mutational inactivation of PTEN does not play a major role in uterine sarcoma tumorigenesis, and another gene or genes on chromosome 10q may be implicated as a cause of these cancers. Differences in the molecular alterations underlying the development of uterine sarcomas and adenocarcinomas are significant.

Favorable survival associated with microsatellite instability in endometrioid endometrial cancers.

OBJECTIVE: To determine whether microsatellite instability in endometrioid endometrial cancer is associated with favorable survival. METHODS: Microsatellite instability analysis was performed in 131 patients with endometrioid endometrial cancer using three polymorphic markers in paired cancer and normal DNA. Logistic regression and multivariable analyses calculated the relation between microsatellite instability, clinical features, and survival. RESULTS: Microsatellite instability was detected in 29 of 131 (22%) endometrioid endometrial cancers. There was no correlation between microsatellite instability and age, race, grade, stage, or depth of myometrial invasion. Microsatellite instability was associated with better survival in univariate and multivariable analyses after controlling for confounding influences (P =.03). The 5-year survival rate of those with microsatellite instability was 77% (95% confidence interval 55%, 90%) compared with only 48% (95% confidence interval 39%, 57%) in other cases. Microsatellite instability was associated with other molecular features that predict favorable outcome including PTEN mutation (P =.002) and the absence of p53 overexpression (P =.01). CONCLUSION: Microsatellite instability is a molecular alteration associated with favorable outcome in endometrioid endometrial cancers, even when accounting for other prognostic factors. This association might be explained by the finding that the pathway of molecular carcinogenesis characterized by loss of DNA mismatch repair favors alteration of genes that result in a less virulent clinical phenotype.

Advances in uterine leiomyoma research: conference overview, summary, and future research recommendations.

Uterine leiomyomas (fibroids, myomas) are the most common tumors occurring in the genital tract of women over 30 years of age. These benign uterine smooth-muscle tumors are estimated to be clinically significant in at least 25% of the American female population during their reproductive years. Furthermore, when thorough pathologic examination of hysterectomy specimens has been performed in patients with or without clinical history of myomatous uteri, the incidence of fibroids is 77%, suggesting that these tumors are far more prevalent than estimated by clinical cases. In spite of their high prevalence, little is known concerning the etiology or the molecular basis of their development and growth. It is well known that leiomyoma growth is regulated by ovarian steroid hormones, yet the exact molecular pathway(s) involved in tumor growth and the role of genetic susceptibility/predisposition and the environment are unclear. This article is an overview of some of the topics addressed at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research. A summary of research needs and recommendations for future research directions based on conference discussions are also presented.

Racial disparity in the frequency of PTEN mutations, but not microsatellite instability, in advanced endometrial cancers.

Survival of African Americans with endometrial cancer is significantly worse than that of whites. Mutation of the PTEN tumor suppressor gene and microsatellite instability occur in some endometrial cancers, and they are associated with favorable prognostic features. The aim of this study was to determine whether there is a racial disparity in the frequency of these molecular alterations that contributes to differences in outcome in advanced endometrial cancer. We screened 140 stage III/IV endometrial adenocarcinomas (78 Caucasian, 62 African American) for mutations in the PTEN gene. Paired DNA samples were available in 100 cases and were analyzed for microsatellite instability using three polymorphic markers. African-American women had cancers with significantly higher stage and grade that were more often nonendometrioid. In addition, median survival of African Americans (1.0 years) was worse than that of whites (2.5 years; P = 0.02). PTEN mutation was seen in 20 of 140 (14%) cancers and was associated with endometrioid histology and more favorable survival. The frequency of PTEN mutations was significantly higher in whites (17 of 78; 22%) than in African Americans (3 of 62; 5%; P = 0.006). Microsatellite instability was found in 15% of cancers, exclusively in endometrioid cases, and was associated with favorable survival (P = 0.01). There was no racial difference in the frequency of microsatellite instability. We conclude that mutation of the PTEN tumor suppressor gene is associated with favorable survival in advanced endometrial cancer and is 4-fold more frequent in Caucasians relative to African Americans. This suggests that differences in the frequency of PTEN mutations contribute to the racial disparity in endometrial cancer survival.

The linoleic acid metabolite, 13-HpODE augments the phosphorylation of EGF receptor and SHP-2 leading to their increased association.

In previous studies with Syrian hamster embryo fibroblasts, we found that a specific lipoxygenase metabolite of linoleic acid, 13(S)-hydroperoxyoctadecadienoic acid (HpODE), enhanced epidermal growth factor (EGF) signal transduction in a tumor suppressor gene plus phenotype (supB+); with a diminished response to 13(S)-HpODE in a tumor suppressor gene minus phenotype (supB-). This differential response was attributed to differences in the rate of EGF receptor (EGFR) dephosphorylation. To further define the molecular basis for these observations, in this report we examine the interaction of phosphorylated EGFR with the SH2 domain-containing protein tyrosine phosphatase, SHP-2, a positive modulator of EGF dependent cell growth. SHP-2 associated with phosphorylated EGFR to a greater extent in supB+ cells when compared to supB-. This differential association could not be accounted for by differences between suppressor gene phenotypes in SHP-2 protein level or mutations in the molecular sequence. The addition of 13(S)-HpODE stimulated a concentration-dependent increase in EGF-dependent phosphorylation of SHP-2 and its association with EGFR. A more dramatic response was observed in the supB+ cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+ and supB- cells. EGFR-SHP-2 association appears to play an important role in the regulation of EGFR signal transduction.

Mutations and altered expression of the human cancer genes: what they tell us about causes.

To understand the causes of cancer, it is necessary to elucidate the molecular basis and environmental factors that influence the carcinogenesis process. Cancers are progressive diseases characterized by the accumulation of defects in many different genes. The patterns of mutation of some genes identified in tumours suggest a direct action of chemicals binding to and altering DNA. Other cancer-associated genes may be altered as a consequence of endogenous mutagens, germ-line mutations, spontaneous mutations that occur during cell replication or increased genetic instability in precancerous cells. Recent advances in molecular biology and genetics have provided new tools and concepts for studying the causes of cancer. We know that cancers are caused by a combination of environmental and genetic factors, and the discovery of the molecular alterations that occur at various stages in different tumours is increasing our understanding of these causes. Thus, we are now beginning to discover which genes are involved, how they function normally and in tumour tissues and why cancers develop after a series of genetic and epigenetic changes in certain cells. As data from studies on cancer-associated genes have accrued, the categories of genes and molecular pathways that have been found to play a role in carcinogenesis have also increased. Genes involved in development and other normal cellular processes have been implicated in cancer. These include genes involved in signal transduction, cell cycle control, DNA repair, cell growth and differentiation (growth factors and growth factor receptors), transcriptional regulation, senescence and apoptosis. Genes involved in angiogenesis, immune regulation, cellular responses to stress, motility, adhesion and invasion are also involved, but less is known about their relationship to carcinogenesis, and these processes are not discussed in this review. The diverse nature of these categories of cancer-related genes indicates the variety of processes that must be disrupted in order for tumours to develop. Many of the genes have several functional domains, and the functions of some have only recently been proposed. In this review, we describe some of the major classes of genes implicated in human cancers and some of the major findings on genetic alterations and dysfunction in human tumours. Comparisons are made with certain rodent models.

Melatonin does not inhibit estradiol-stimulated proliferation in MCF-7 and BG-1 cells.

Melatonin, an indolic pineal hormone, is produced primarily at night in mammals and is important in controlling biological rhythms. Previous research suggested that melatonin can attenuate proliferation in the estrogen-responsive MCF-7 breast cancer cell line. We tested whether these anti-proliferative effects may have physiological consequences upon two estrogen-responsive cell lines, MCF-7 (a breast cancer cell line) and BG-1 (an ovarian adenocarcinoma cell line). Melatonin (10(-9)-10(-5) M) attenuated proliferation of MCF-7 and BG-1 cells by >20% in the absence of estrogen. However, 17beta-estradiol exposure negated the ability of melatonin to inhibit proliferation. To substantiate this finding, cells were estrogen starved followed by multiple treatments with estradiol and melatonin. Melatonin did not inhibit estradiol-stimulated proliferation under this protocol. Estradiol increased MCF-7 and BG-1 cell cycle transition from G1 to S phase, however, melatonin did not inhibit this transition nor did it down-regulate estradiol-induced pS2 mRNA levels measured by northern blotting, further indicating that melatonin was unable to attenuate estradiol-induced proliferation and gene expression. We also examined the effects of melatonin on estradiol-induced proliferation in MCF-7 cell xenografts in athymic nude mice. Melatonin at a dose 28 times greater than 17beta-estradiol did not inhibit estradiol-induced proliferation in vivo. Furthermore, pinealectomy did not increase proliferation. Therefore, we conclude that melatonin does not directly inhibit estradiol-induced proliferation.

Resistance to 6-thioguanine in mismatch repair-deficient human cancer cell lines correlates with an increase in induced mutations at the HPRT locus.

Although the resistance to the cytotoxic response of certain DNA damaging agents has been well characterized in cells deficient in mismatch repair, little is known about how such resistance affects mutagenesis. Using human cancer cell lines defective in mismatch repair (MMR) and complementary cell lines in which the MMR defects were corrected by chromosome transfer, we present the cytotoxic effect and the mutagenic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus following exposure to the chemotherapeutic agent, 6-thioguanine (6-TG). Upon exposure to 6-TG, there was a differential cytotoxic response. The MMR-deficient cells were resistant to 6-TG exposure up to 5 microM, whereas the MMR-proficient cell lines were significantly more sensitive at the same levels of exposure. Furthermore, the mutagenic response at HPRT induced by 6-TG was substantially increased in the MMR-deficient lines relative to the MMR-proficient cell lines. These findings support the notion that cytotoxicity to 6-TG is mediated through functional MMR and that resistance to the cytotoxic effects of 6-TG is directly associated with an increase in induced mutations in MMR-defective cells. These data suggest that the use of 6-TG as a chemotherapeutic agent may result in the selection of MMR-defective cells, thereby predisposing the patient to an increased risk for developing secondary tumors.

Allelotype analysis of uterine leiomyoma: localization of a potential tumor suppressor gene to a 4-cM region of chromosome 7q.

Uterine leiomyoma is a benign smooth muscle tumor of the myometrium and is the most commonly encountered neoplasm in women of reproductive age. As for most benign tumors, the pathogenesis of leiomyoma remains obscure, especially at the molecular genetic level. The purpose of this study was to perform a genome-wide allelotype analysis to identify potential sites of tumor suppressor gene inactivation. Fifty-two cases of uterine leiomyoma were subjected to allelotype analysis by using matched pairs of tumor and blood DNA. Loss of heterozygosity (LOH) was assessed at 61 microsatellite markers distributed throughout the genome and representing all 41 chromosome arms. In general, LOH was very rare except on chromosome 7q, where LOH was observed in 34% of all informative tumors. Fine-deletion mapping with 25 microsatellite markers from the 7q22 region revealed a minimal deletion unit of approximately 4 cM, bounded by the markers D7S2453 proximally and D7S496 distally, that probably harbors a novel tumor suppressor gene involved in the etiology of this tumor.

PTEN mutation in endometrial cancers is associated with favorable clinical and pathologic characteristics.

Mutation of the PTEN tumor suppressor gene is a frequent event in endometrial cancers. In other types of cancers, PTEN mutation has been associated with metastatic behavior and advanced stage. To examine the relationship between PTEN mutation and clinical features of endometrial cancers, we screened 136 cases for mutations in the nine exons and intronic splice sites of the PTEN gene, using single-strand conformation analysis, and aberrant bands were sequenced. Mutations were noted in 44 of 136 (32%) endometrial cancers, and two mutations were present in 8 cases. There were 36 cases with mutations resulting in truncated protein products, 6 cases with missense mutations in the phosphatase domain, 1 case with an in-frame deletion, and 1 case with a large insertion. Mutation of the PTEN gene correlated most closely with endometrioid histology; mutations were seen in only 5% (1 of 21) of serous/clear cell cancers compared with 37% (43 of 115) of endometrioid cancers (P = 0.004). PTEN mutation was associated with early stage, nonmetastatic disease and more favorable survival in both the entire group of 136 cases and in the 115 endometrioid cases. In addition, PTEN mutation correlated with other molecular features associated with favorable clinical behavior, including microsatellite instability and absence of p53 overexpression. Microsatellite instability was found in 60% of cases with PTEN mutations compared with only 25% of cases without mutations (P = 0.004). Overexpression of p53 was seen in only 14% of cases with PTEN mutations compared to 39% of cases without mutations (P = 0.006). In conclusion, PTEN mutation is associated with endometrioid histology and other favorable pathological, clinical, and molecular features rather than with increased metastatic potential as has been noted in some other types of cancers.

Stability of microsatellites in myeloid neoplasias.

Microsatellites are short, repeated DNA sequences that exist throughout the genome. Instability of these sequences, associated with defects in the DNA mismatch repair system, is the hallmark of hereditary non-polyposis colorectal cancer (HNPCC), and is also found in many sporadic cancers. Although many types of solid tumors exhibit this type of genetic instability, its involvement in hematologic cancers is less evident. We have investigated whether microstatellite instability (MSI) is involved in the transformation of myeloid cells to myelodysplastic syndrome (MDS) and/or acute myelogenous leukemia (AML). Both de novo and treatment-associated neoplasias were studied. Only one example of MSI was found in 48 patients, using a panel of 14 different microsatellite loci consisting of repeats of one to four base pairs. These results suggest that the genes responsible for MSI are not involved in the transformation of normal myeloid cells to MDS or AML.

Characterization of distinct human endometrial carcinoma cell lines deficient in mismatch repair that originated from a single tumor.

The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in the hMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6 locus. However, HEC-1-B cells are heterozygous at the hPMS2 locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer, with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1-A cells, illustrating the repair of frameshift intermediates by hMSH2/hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells. These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer.

BG-1 ovarian cell line: an alternative model for examining estrogen-dependent growth in vitro.

Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17Beta-estradiol, epidermal growth factor, and insulin-like growth factorinduced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa > MDA-MB-435 > HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.

The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts.

Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.

Mutation of the PTEN tumor suppressor gene is not a feature of ovarian cancers.

OBJECTIVE: The PTEN tumor suppressor gene on chromosome 10q23 undergoes inactivating mutations in several types of malignancies including glioblastomas and prostate and endometrial carcinomas. The aim of this study was to determine if mutation of the PTEN tumor suppressor gene is a feature of sporadic or BRCA1-associated ovarian carcinomas. METHODS: Genomic deoxyribonucleic acid was extracted from 11 ovarian cancer cell lines and 50 frozen ovarian cancers, including 4 cases that developed in women with germline mutations in the BRCA1 breast/ovarian cancer susceptibility gene. The polymerase chain reaction was used to amplify each of the nine exons and intronic splice sites of the PTEN gene. These products were then screened for mutations using single strand conformation polymorphism analysis. Variant bands were further evaluated using automated DNA sequencing. RESULTS: A previously unreported silent polymorphism at codon 240 (TAT to TAC) in exon 7 was noted in one of the primary ovarian carcinomas. Mutations in the PTEN gene were not found in any of the 50 primary ovarian cancers or 11 immortalized ovarian cancer cell lines. CONCLUSION: Alteration of the PTEN tumor suppressor gene does not appear to be a feature of sporadic or BRCA1-associated ovarian cancers.

Single gene complementation of the hPMS2 defect in HEC-1-A endometrial carcinoma cells.

Results from the analysis of human tumor cell lines with mutations in DNA mismatch repair genes have contributed to the understanding of the functions of these gene products in DNA mismatch repair, microsatellite instability, cell cycle checkpoint control, transcription-coupled nucleotide excision repair, and resistance to cytotoxic agents. However, complementation of human DNA mismatch repair defects by introduction of a single cloned gene or cDNA, which would serve to directly prove or disprove their involvement in these processes, has not been accomplished. Here, we introduce a wild-type copy of the hPMS2 cDNA by stable transfection into the PMS2 mutant HEC-1-A cell line. HEC-1-A cells expressing wild-type hPMS2 exhibit increased microsatellite stability, have a reduced mutation rate at the endogenous hypoxanthine phosphoribosyltransferase locus and extracts from these cells are able to perform strand-specific mismatch repair. These results demonstrate that the hPMS2 gene is integral to the maintenance of genome stability.