Expression of DCC and netrin-1 in normal human endometrium and its implication in endometrial carcinogenesis

OBJECTIVE: Although DCC has been considered as a candidate tumor suppressor, the roles it plays in the uterine endometrium and in the carcinogenic process remains unclear. To define these roles more clearly, we examined the expression of DCC and its ligand, netrin-1, in the normal endometrium and in endometrial cancer. METHODS: The expression of DCC and netrin-1 in normal endometrial glands and in cancer cell lines was examined by RT-PCR and immunohistochemistry. The effects of exogenous DCC and netrin-1 expression were observed together with the respective expression vector transfection. RESULTS: Endometrial glands in the proliferative and early secretory phase expressed both DCC and netrin-1, but glands in the late-secretory phase tended to silence DCC expression. In addition, all of the endometrial cancer cell lines lost normal DCC expression. Restored DCC expression in the cancer cell lines in the absence of netrin-1 induced apoptosis. However, no changes were observed in the presence of netrin-1. CONCLUSION: Our observations suggest that DCC/netrin-1 signaling may commit cells to the transition of endometrial gland architecture or function from a proliferating to a secretory phase. In addition, the silencing of DCC expression may contribute to the escape of endometrial cancer cells from a DCC-regulated apoptotic program, thereby promoting malignant phenotypes.     

Gestational choriocarcinoma whose responsible pregnancy was a complete hydatidiform mole identified by PCR analysis with new sequence tagged site primers

We report a case where the pregnancy responsible for a gestational choriocarcinoma was not the antecedent pregnancy or the second normal term delivery, but a complete hydatidiform mole that had advanced to clinically invasive mole. This responsible pregnancy was identified by polymerase chain reaction analysis (PCR). PCR analysis was performed by using five new sets of sequence-tagged site (STS) primers on four chromosomes (chr. 1, D1S225; chr. 3, D3S1744; chr. 12, D12S1090; chr. 18, D18S849 and D18S877). The constitution of alleles of choriocarcinoma was shown to be almost identical with that of the husband on every marker. The allele patterns of choriocarcinoma on D3S1744 and D12S1090 were not observed with DNA from the patient. The band pattern originating from molar DNA was also identical with those of the husband and choriocarcinomas on D18S849 and D1S225.     

FBXW7 is involved in the acquisition of the malignant phenotype in epithelial ovarian tumors

FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, such as c‐Myc, cyclin E, Notch and c‐Jun. FBXW7 is a known tumor‐suppressor gene, and mutations in FBXW7 have been reported in various human malignancies. In this study, we examined the sequences of the FBXW7 and p53 genes in 57 ovarian cancer clinical samples. Interestingly, we found no FBXW7 mutations associated with amino acid changes. We also investigated FBXW7 expression levels in 126 epithelial ovarian tumors. FBXW7 expression was negatively correlated with the malignant potential of ovarian tumors. That is to say, FBXW7 expression levels in ovarian cancer samples were significantly lower than those in borderline and benign tumors (P < 0.01). FBXW7 expression levels in serous carcinoma samples were the lowest among four major histological subtypes. In addition, p53‐mutated ovarian cancer samples showed significantly lower levels of FBXW7 expression compared with p53 wild‐type cancer samples (P < 0.001). DNA methylation arrays and bisulfite PCR sequencing experiments revealed that 5′‐upstream regions of FBXW7 gene in p53‐mutated samples were significantly higher methylated compared with those in p53 wild‐type samples (P < 0.01). This data indicates that p53 mutations might suppress FBXW7 expression through DNA hypermethylation of FBXW7 5′‐upstream regions. Thus, FBXW7 expression was downregulated in ovarian cancers, and was associated with p53 mutations and the DNA methylation status of the 5′‐upstream regions of FBXW7.     

An inhibitory effect on cell proliferation by blockage of the MAPK/estrogen receptor/MDM2 signal pathway in gynecologic cancer

OBJECTIVE: We previously demonstrated that that the Ras/ER/MDM2 pathway was critical for NIH3T3 cell transformation. In this study, we examined the effect of blocking this pathway on cell growth in gynecologic cancer cells. METHODS: (1) The levels of MDM2, ER, p53 and p21 in endometrial or ovarian cancer cell lines were investigated and compared with that in normal cells by Western blots. (2) The effects of MEK-inhibitor and/or anti-estrogen, and siRNA of MDM2 on cell growth, tumorigenicity in nude mice were examined. RESULTS: The MDM2 level was enhanced in cancer cells compared with normal cells. Treatment with MEK inhibitor(U0126) resulted in a reduced MDM2 level, enhanced p53 and p21 levels and inhibited cell growth by the induction of premature senescence. The effect of MEK inhibitor on cell growth was affected by ER levels and functions. Treatment with low-dose MEK inhibitor in combination with anti-estrogen (ICI182,780) had a more inhibitory effect on cell growth compared to treatment with MEK inhibitor or anti-estrogen alone in cancer cells. Down-regulation of the MDM2 level by siRNA resulted in the inhibition of growth in cancer cells. CONCLUSION: The blockage of the MAPK/ER/MDM2 pathway suppress cell proliferation and it is supposed as a new molecular target therapy in estrogen-dependent gynecologic cancers, such as endometrial or ovarian cancer.     

Three‐dimensional power Doppler transanal ultrasonography,to monitor haemorrhoidal blood flow after Doppler‐guided ALTA sclerosing therapy

Aim The study aimed to use power Doppler imaging (PDI) transanal ultrasonography to produce three‐dimensional power Doppler angiography images of haemorrhoidal tissue and to monitor the effects of Doppler‐guided aluminium potassium sulfate and tannic acid (DGALTA) sclerotherapy. Method Ninety‐six haemorrhoids in 43 patients were examined using PDI transanal ultrasonography, and DGALTA sclerotherapy was performed from April 2011 to April 2012. DGALTA sclerotherapy was conducted using a four‐step injection process with pulse wave Doppler ultrasound under perianal local anaesthesia. Results A three‐dimensional power Doppler angiography image of the blood flow in haemorrhoidal tissue was produced using PDI transanal ultrasonography. The cross‐sectional area of blood flow in the haemorrhoidal tissue (PDI area) significantly decreased after DGALTA sclerotherapy. The PDI areas in the preoperative state and 1 and 3 months after treatment were 0.35 ± 0.27, 0.03 ± 0.05 and 0.04 ± 0.05 cm² (P < 0.0001). Conclusion A three‐dimensional power Doppler angiography image of the haemorrhoidal tissue was technically possible and showed blood flow in the haemorrhoidal tissue to be significantly decreased after DGALTA sclerotherapy.     

Level of reactive oxygen species induced by p21WAF(1)/CIP(1) 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, γH2AX, 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. ( Cancer Sci 2009; 100: 1275–1283)