Association of MDM2 SNP309 and TP53 Arg72Pro polymorphisms with risk of endometrial cancer.

The incidence of endometrial cancer, a common gynecological malignancy, is increasing in Japan. We have previously shown that the ER/MDM2/p53/p21 pathway plays an important role in endometrial carcinogenesis. In the present study, we investigated the effects of germline single nucleotide polymorphisms in murine double minute 2 (MDM2) SNP309, TP53 Arg72Pro, ESR1 PvuII and XbaI, and p21 codon 31 on endometrial cancer risk. We evaluated these polymorphisms in DNA samples from 125 endometrial cancer cases and 200 controls using polymerase chain reaction-based restriction fragment length polymorphism. The association of each genetic polymorphism with endometrial cancer was examined by the odds ratio and 95% confidence interval, which were obtained using logistic regression analysis. The SNP309 GG genotype non-significantly increased the risk of endometrial cancer. The 95% confidence interval for the GG genotype vs. the TT genotype of MDM2 SNP309 was 1.76 (0.93-3.30). Endometrial cancer was not associated with tested SNP genotypes for TP53, ESR1 and p21. The combination of SNP309 GG + TG and TP53 codon 72 Arg/Arg significantly increased endometrial cancer risk. The adjusted OR was 2.53 (95% confidence interval, 1.03-6.21) and P for the interaction was 0.04. This result was supported by in vitro data showing that endometrial cancer cell lines with the SNP309 G allele failed to show growth inhibition by treatment with RITA, which reduces p53-MDM2 binding. The presence of the SNP309 G allele and TP53 codon 72 Arg/Arg genotype is associated with an increased risk of endometrial cancer in Japanese women.

Sodium butyrate inhibits the self-renewal capacity of endometrial tumor side-population cells by inducing a DNA damage response.

We previously isolated side-population (SP) cells from a human endometrial cancer cell line, Hec1, and determined that Hec1-SP cells have cancer stem-like cell features. In this study, we isolated SP cells and non-SP (NSP) cells derived from a rat endometrial cell line expressing human [(12)Val] KRAS (RK12V cells) and determined the SP phenotype. RK12V-SP cells showed self-renewal capacity, the potential to develop into stromal cells, reduced expression levels of differentiation markers, long-term proliferating capacity in cultures, and enhanced tumorigenicity, indicating that RK12V-SP cells have cancer stem-like cell features. RK12V-SP cells also display higher resistance to conventional chemotherapeutic drugs. In contrast, treatment with a histone deacetylases (HDAC) inhibitor, sodium butyrate (NaB), reduced self-renewal capacity and completely suppressed colony formation of RK12V-SP cells in a soft agar. The levels of intracellular reactive oxygen species (ROS) and the number of γH2AX foci were increased by NaB treatment of both RK12V-SP cells and RK12V-NSP cells. The expression levels of γH2AX, p21, p27, and phospho-p38 mitogen-activated protein kinase were enhanced in RK12V-SP cells compared with RK12V-NSP cells. These results imply that treatment with NaB induced production of intracellular ROS and DNA damage in both RK12V-SP and RK12V-NSP cells. Following NaB treatment, DNA damage response signals were enhanced more in RK12V-SP cells than in RK12V-NSP cells. This is the first article on an inhibitory effect of NaB on proliferation of endometrial cancer stem-like cells. HDAC inhibitors may represent an attractive antitumor therapy based upon their inhibitory effects on cancer stem-like cells.

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation.

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 µM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.

Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage.

Cancer stem-like cell subpopulations, referred to as "side-population" (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, alpha-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into alpha-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.

Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.

p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.

Induction of senescence by progesterone receptor-B activation in response to cAMP in ovarian cancer cells.

OBJECTIVE: Progesterone receptor (PR) expression is a favorable prognostic marker in ovarian cancer. We previously demonstrated that the induction of PR-B by treatment with cAMP was associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH 3T3 cells. In this study, we examined the effect of cAMP treatment on cell growth in Ras-transformed NIH3T3 cells and ovarian cancer cells. METHODS: 1) The levels of PR-B and cell cycle associated proteins (p21, p27 and Rb) following treatment with cAMP in the Ras-transformed NIH3T3 cells (K12V) and ovarian cancer cell lines (SKOV cells) were investigated by Western blots. 2) The effects of PR overexpression following treatment with cAMP or after infection of an adenovirus expressing PR-B on cell growth and tumorigenicity in a soft agar culture were examined. RESULTS: 1) Treatment with cAMP increased PR-B and p27 levels in K12V cells and inhibited cell growth by inducing premature senescence. Induction of senescence was specific to the transformed cells. 2) In SKOV cells, treatment with cAMP induced PR-B, p27 and p21 expression, reduced the level of phosphorylated Rb, caused accumulation of cells in the G0/G1 fraction of the cell cycle, and induced senescence. 3) Both anchorage-dependent and -independent SKOV cell growths were inhibited by cAMP treatment. 4) Induction of both the expression and transcriptional activity of PR-B is critical for the induction of senescence and suppression of tumorigenicity. CONCLUSION: Treatment of cAMP, through activation of PR-B, induced senescence and suppressed tumorigenicity in ovarian cancer cells.