Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family

Members of the Bcl-2 family of apoptosis-regulating proteins contain at least one of the four evolutionarily conserved domains, termed BH1, BH2, BH3, or BH4. Here, we report the identification, cloning, physical mapping, and expression pattern of BCL2L12, a novel gene that encodes a BCL2-like proline-rich protein. Proline-rich sites have been shown to interact with Src homology region 3 (SH3) domains of several tyrosine kinases, mediating their oncogenic potential. This new gene maps to chromosome 19q13.3 and is located between the IRF3 and the PRMT1/HRMT1L2 genes, close to the RRAS gene. BCL2L12 is composed of seven coding exons and six intervening introns, spanning a genomic area of 8.8 kb. All of the exon-intron splice sites conform to the consensus sequence for eukaryotic splice sites. The BCL2L12 protein is composed of 334 amino acids, with a calculated molecular mass of 36.8 kDa and an isoelectric point of 9.45. The BCL2L12 protein contains one BH2 homology domain, one proline-rich region similar to the TC21 protein and, five consensus PXXP tetrapeptide sequences. BCL2L12 is expressed mainly in breast, thymus, prostate, fetal liver, colon, placenta, pancreas, small intestine, spinal cord, kidney, and bone marrow and to a lesser extent in many other tissues. We also identified one splice variant of BCL2L12 that is primarily expressed in skeletal muscle.     

Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

Membranous levels of c-erbB-2 oncoprotein in gastric cancer: their relationship with clinicopathological parameters and their prognostic significance

BACKGROUND: The protein encoded by the c-erbB-2 gene is a membrane receptor expressed in a variety of solid human cancers and directly related to poor prognosis. The objective of this work was to evaluate the clinical value of the quantification of membranous oncoprotein levels in gastric cancer. MATERIALS AND METHODS: Membranous c-erbB-2 levels were examined by means of a sandwich immunoenzymatic assay in 82 patients with gastric cancer. The median follow-up period for these patients was 16 months. In addition, c-erbB-2 expression was analyzed by immunohistochemistry in 57 gastric carcinomas. RESULTS: Membranous c-erbB-2 levels ranged widely in the studied tumors (44-112,000 NHU/mg protein). Median c-erbB2 content was significantly higher in intestinal-type tumors than in diffuse-type tumors (p = 0.01). In addition, high levels of c-erbB-2 were significantly associated with shorter relapse-free survival and overall survival in patients with resectable gastric carcinomas (p = 0.01 and p = 0.04, respectively). However, the correlation between immunohistochemistry and ELISA determinations did not reach statistical significance. CONCLUSION: Our results suggest a potential prognostic value of membranous c-erbB-2 quantification by immunoenzymatic assay in gastric cancer. However, its possible role in the selection of patients with a view to the possible introduction of Herceptin as a novel drug against gastric cancer is at present uncertain.     

LIN28 Is Involved in Glioma Carcinogenesis and Predicts Outcomes of Glioblastoma Multiforme Patients

LIN28, an evolutionarily conversed RNA binding protein which can bind to the terminal loops of let-7 family microRNA precursors and block their processing to maturation, is highly expressed in several subsets of tumors that carry poor prognoses, such as ovarian carcinoma, hepatocellular carcinoma, colon carcinoma and germ cell carcinoma. However, there has been no study on the expression of LIN28 in glioma tissues or their importance as a prognostic predictor of glioma patients. This study aimed to examine the expression of LIN28 in glioma and correlate the results to patient outcome. We found that LIN28 expression was significantly higher in the group of patients with a poor prognosis compared to patients with a good prognosis by gene microarray. Log-rank analysis showed patients with higher LIN28 expression level in tumor had a shorter progression-free survival and overall survival times compared to those with lower LIN28 expression level. Similar results were also obtained from the tissue microarray analysis. Univariate and multivariate analyses showed high LIN28 expression was an independent prognostic factor for a shorter progression-free survival and overall survival in GBM patients. Furthermore in vitro experiments showed that down-regulation of LIN28 in U251 and U373 cells caused cell cycle arrest in the G1 phase, delayed cell proliferation, increased apoptosis, and resulted in fewer colonies compared to controls. Summarily, our data provides a potential target for cancer therapy as an approach to overcome the poor options currently available for GBM patients.     

Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor

The ING1 gene encodes p33(ING1), a putative tumor suppressor for neuroblastomas and breast cancers, which has been shown to cooperate with p53 in controlling cell proliferation. We have isolated a novel human gene, ING1L, that potentially encodes a PHD-type zinc-finger protein highly homologous to p33(ING1). Fluorescence in situ hybridization and radiation-hybrid analyses assigned ING1L to human chromosome 4. Both ING1 and ING1L are expressed in a variety of human tissues, but we found ING1L expression to be significantly more pronounced in tumors from several colon-cancer patients than in normal colon tissues excised at the same surgical sites. Although the significance of this observation with respect to carcinogenesis remains to be established, the data suggest that ING1L might be involved in colon cancers through interference with signal(s) transmitted through p53 and p33(ING1).     

LncRNA HAND2-AS1 exerts anti-oncogenic effects on ovarian cancer via restoration of BCL2L11 as a sponge of microRNA-340-5p

In the early stage of ovarian cancer (OC), molecular biomarkers are critical for its diagnosis and treatment. Nevertheless, there is little research on the mechanism underlying tumorigenesis in OC. Herein, we aimed to explore whether long noncoding RNA (lncRNA) HAND2-AS1 participated in the regulation of the cell proliferation, migration, and apoptosis of OC by regulating B-cell lymphoma 2 like 11 (BCL2L11) and microRNA-340-5p (miR-340-5p). Differentially expressed lncRNAs in OC were screened by microarray-based analysis. HAND2-AS1, BCL2L11, and miR-340-5p expression was assessed in normal ovarian and OC tissues and human OC cell lines. Then, the relationships among HAND2-AS1, BCL2L11, and miR-340-5p were explored. Ectopic expression and depletion experiments were applied to analyze the effects of HAND2-AS1, miR-340-5p and BCL2L11 on migration, invasion, and proliferation of OC cells, as well as apoptosis. Lastly, the tumor xenograft in nude mice was conducted to test the tumorigenesis in vivo. In silico analysis displayed poor expression of HAND2-AS1 in OC. HAND2-AS1 specifically sponged with miR-340-5p which was found to directly target BCL2L11. Importantly, HAND2-AS1 or BCL2L11 overexpression or miR-340-5p downregulation resulted in reduction of cell invasion and migration, together with decrease of cell proliferation and increase of cell apoptosis in OC. Besides, high-expressed HAND2-AS1 inhibited the tumorigenesis in nude mice. To sum up, these data suggests HAND2-AS1 as an anti-oncogene in OC through upregulation of BCL2L11 by competitively binding to miR-340-5p, which demonstrates that there are potential diagnosis and therapy values of HAND2-AS1 in OC.     

Expression of SSX-2 and SSX-4 genes in neuroblastoma

The SSX genes are members of the family of cancer/testis antigens that encode tumor-associated antigens recognizable by autologous cytolytic T lymphocytes. Their expression is common in tumors of diverse lineages and absent in normal tissues except testis and thyroid. In this study, sixty-seven neuroblastomas (NB) (12 stage 1, 13 stage 2, 12 stage 3, 12 stage 4S and 13 stage 4) were examined by RT-PCR and a sensitive chemiluminescent detection method for SSX-2 and SSX-4 expression. Seventy-two percent (13/18) of stage 4 NB expressed SSX-2 and 67% (12/18) expressed SSX-4. SSX-2 and SSX-4 positivity correlated with metastatic NB stage 4 (p=0.02 and p=0.006, respectively). Sensitivity experiments showed SSX-2 detection was one tumor cell in 10(6) normal cells, and one in 10(4) for SSX-4. All normal tissues (n=6), with the exception of testis, normal bone marrow (BM, n=12) and normal peripheral blood (PBL, n=10) were negative for SSX-2 and SSX-4 expression. Thirty-two BM and 14 PBL obtained from 35 stage 4 NB patients at 24 months from their diagnosis were evaluated for SSX-2 expression. Unlike another cancer/testis antigen, GAGE, only one BM sample was positive, and no prognostic utility could be established. Further investigation of SSX expression at other relevant time points is warranted.     

Treatment of MCF-7 cells with taxol and etoposide induces distinct alterations in the expression of apoptosis-related genes BCL2, BCL2L12, BAX, CASPASE-9 and FAS

We studied alterations in the mRNA expression levels of BCL2 (Bcl-2), BCL2L12, BAX, FAS and CASPASE-9 genes in the MCF-7 breast cancer cell line in response to treatment with two anticancer drugs. Cell toxicity was evaluated by the MTT method, trypan blue staining and DNA laddering, whereas the expression levels of the apoptosis-related genes were analysed by RT-PCR using gene-specific primers. In the case of etoposide, down-regulation of the BCL2L12-A gene variant and of CASPASE-9, as well as upregulation of BAX, was observed, whereas treatment of MCF-7 cells with taxol led to down-regulation of the mRNA levels of all genes examined. Our results support the idea that after long-term clinical studies, mRNA expression analysis of BCL2L12 and other members of the BCL2 gene family may serve as useful molecular markers predicting chemotherapy response in breast cancer.