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.     

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.     

Cisplatin downregulates BCL2L12, a novel apoptosis-related gene, in glioblastoma cells

Glioblastoma progression is mainly characterized by intense apoptosis resistance and marked necrosis. Over-expression of BCL2L12, a novel member of Bcl-2 family has been shown in primary glioblastoma. BCL2L12 blocks effective caspase-3/7 maturation and inhibits p53 tumor suppressor, deriving resistance toward apoptosis and inducing extensive cell necrosis. Cisplatin is a major chemotherapeutic agent which has a broad range of anti-neoplastic activities including apoptosis induction. To investigate the effect of cisplatin on the expression of BCL2L12 in glioblastoma cells, two glioblastoma cell lines were treated with different concentrations of cisplatin for 48 h. The cell viability and IC50 was determined using MTT assay. Then, the two glioblastoma cell lines were treated with 48 h IC50 concentration of cisplatin for 24, 48, and 72 h. Apoptosis induction was analyzed by fluorescence microscopy and flow cytometry. Gene expression study was performed on BCL2L12 and TBP as target and internal control genes, respectively. The quantitative real-time polymerase chain reaction results showed that BCL2L12 gene expression was significantly (p?=?0.001) downregulated in the presence of cisplatin. In conclusion, cisplatin treatment induced a time-dependent apoptosis in glioblastoma cells, at least partially via downregulation of BCL2L12 gene expression.     

Molecular profile of breast versus ovarian cancer cells in response to treatment with the anticancer drugs cisplatin, carboplatin, doxorubicin, etoposide and taxol

We assessed changes in the apoptosis-related genes BCL2, BAX, BCL2L12, FAS and CASPASE-3 in OVCAR-3 human ovarian cancer cells and BT-20 human breast cancer cells to provide an insight into the molecular mechanisms involved in the response of these cells to treatment with anticancer drugs and to assess their value as potential biomarkers of chemotherapy response in breast and ovarian cancer. Cells were treated with different chemotherapeutic drugs (cisplatin, carboplatin, doxorubicin, etoposide and taxol) and assessed for changes in the expression of apoptosis-related genes at the mRNA level. Total RNA was extracted, reverse-transcribed into cDNA and amplified by PCR using gene-specific primers. GAPDH was used as a housekeeping gene. Cytotoxicity was assessed by MTT assay. Both cancer cell lines responded differentially at the molecular level to the drug treatments. OVCAR-3 cells showed more pronounced sensitivity and changes compared to BT-20 cells at the mRNA level for different apoptosis-related genes, leading to cell and cancer type dependence in conjunction with drug dependence.     

GSK3β regulates Bcl2L12 and Bcl2L12A anti-apoptosis signaling in glioblastoma and is inhibited by LiCl

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b binding partner in a testis cDNA library. Our data demonstrated that GSK3b interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12_153–191 fragment located outside of the C-terminal BH2 motif is responsible for GSK3b binding. In contrast, no interaction was detected between BCL2L12A and GSK3b. In vitro kinase and l-phosphatase assays showed that GSK3b phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12_153–191 fragment directly interrupted GSK3bmediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3b interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.     

L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers

Aberrant beta-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of beta-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.