Functional Characterization of CLPTM1L as a Lung Cancer Risk Candidate Gene in the 5p15.33 Locus

Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy

Cell Free Expression of hif1α and p21 in Maternal Peripheral Blood as a Marker for Preeclampsia and Fetal Growth Restriction

Preeclampsia, a severe unpredictable complication of pregnancy, occurs in 6% of pregnancies, usually in the second or third trimester. The specific etiology of preeclampsia remains unclear, although the pathophysiological hallmark of this condition appears to be an inadequate blood supply to the placenta. As a result of the impaired placental blood flow, intrauterine growth restriction (IUGR) and consequential fetal oxidative stress may occur. Consistent with this view, pregnancies complicated by preeclampsia and IUGR are characterized by up-regulation of key transcriptional regulators of the hypoxic response including, hif1α and as well as p53 and its target genes. Recently, the presence of circulating cell-free fetal RNA has been documented in maternal plasma. We speculated that pregnancies complicated by preeclampsia and IUGR, will be associated with an abnormal expression of p53 and/or hif1α related genes in the maternal plasma. Maternal plasma from 113 singleton pregnancies (72 normal and 41 complicated pregnancies) and 19 twins (9 normal and 10 complicated pregnancies) were collected and cell free RNA was extracted. The expression of 18 genes was measured by one step real-time RT-PCR and was analyzed for prevalence of positive/negative expression levels. Results indicate that, among the genes examined, cell free plasma expressions of p21 and hif1α were more prevalent in pregnancies complicated by hypoxia and/or IUGR (p<0.001). To conclude, we present in this manuscript data to support the association between two possible surrogate markers of hypoxia and common complications of pregnancy. More work is needed in order to implement these findings in clinical practice

BRCA1 Regulates Follistatin Function in Ovarian Cancer and Human Ovarian Surface Epithelial Cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells

Next-Generation Sequencing of Apoptotic DNA Breakpoints Reveals Association with Actively Transcribed Genes and Gene Translocations

DNA fragmentation is a well-recognized hallmark of apoptosis. However, the precise DNA sequences cleaved during apoptosis triggered by distinct mechanisms remain unclear. We used next-generation sequencing of DNA fragments generated in Actinomycin D-treated human HL-60 leukemic cells to generate a high-throughput, global map of apoptotic DNA breakpoints. These data highlighted that DNA breaks are non-random and show a significant association with active genes and open chromatin regions. We noted that transcription factor binding sites were also enriched within a fraction of the apoptotic breakpoints. Interestingly, extensive apoptotic cleavage was noted within genes that are frequently translocated in human cancers. We speculate that the non-random fragmentation of DNA during apoptosis may contribute to gene translocations and the development of human cancers