Raj P. Kandpal

EphB6 receptor significantly alters invasiveness and other phenotypic characteristics of human breast carcinoma cells

Breast cancer mortality in women is largely attributed to the metastasis of primary breast tumors. We have analysed the function of EphB6, a kinase-deficient receptor, in the invasive phenotype of breast cancer cell lines. We have demonstrated the loss of EphB6 protein in invasive breast carcinoma cell lines and absence of EphB6 transcript in a metastatic breast tumor specimen. The function of EphB6 in invasiveness was confirmed by the ability of EphB6 protein to decrease the in vitro invasiveness of MDA-MB-231, MDA-MB-435 and BT549 cells transfected with an EphB6 expression construct. In MDA-MB-231 cells, the decreased invasiveness appeared to be mediated by decreased transcript levels of matrix metalloproteinase (MMP)7 and MMP19, and increased transcript levels of tissue inhibitors of metalloproteinase 2. In addition to affecting invasiveness phenotype, EphB6 overexpression was also responsible for altering the growth rate and colony-forming efficiency of MCF-7 and MDA-MB-231 cells in a cell-line-specific manner. We suggest that the significant decrease in the invasiveness of MDA-MB-231 and other cell lines transfected with EphB6 is likely occurring by the ability of EphB6 to transduce signals to the nucleus and altering relevant gene expression.

EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

Caveolin-1 and Flotillin-1 Differential Expression in Clinical Samples of Renal Cell Carcinoma

Caveolin-1 and flotillin-1 belong to plasma membrane microdomains. They are characterized by peculiar lipid and protein composition and are involved in fundamental cellular events such as: signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 30 human renal cell carci- noma (RCC) and adjacent normal kidney (ANK) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 over-expression was found in RCC tissues compared to ANK, and was confirmed by immunohistochemistry. Caveolin-1 and flotillin-1 protein levels were found by 1-D, 2-DE, and MS to be increased also in RCC microdomain-enriched subcellular fractions purified from paired RCC and ANK samples.

Chromosome 6 encoded RNaseT2 protein is a cell growth regulator.

We have previously shown by chromosome transfer technique that chromosome 6 alters the phenotype of a variety of tumour cells and SV40 immortalized cells. We present here the phenotypic effects of the ectopic expression of RNaseT2, a highly conserved ribonuclease encoded by chromosome 6q27, in SV40 immortalized cell lines. We contrast our findings with those reported for ovarian carcinoma cell lines and an SV40 immortalized cell line transfected with RNaseT2. Although RNaseT2 expression is elevated in normal diploid fibroblasts approaching senescence (passage 64), forced expression of the gene in immortalized cells does not cause them to senesce. A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2. The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2–10‐folds in SV40 immortalized cells in response to RNaseT2 expression. Interestingly, some immortalized cells expressed alternatively spliced transcript variants instead of the full‐length RNaseT2 transcript. Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.

Genetic components in diabetic retinopathy

Diabetic retinopathy (DR) is a serious complication of diabetes, which is fast reaching epidemic proportions worldwide. While tight glycemic control remains the standard of care for preventing the progression of DR, better insights into DR etiology require understanding its genetic basis, which in turn may assist in the design of novel treatments. During the last decade, genomic medicine is increasingly being applied to common multifactorial diseases such as diabetes and age-related macular degeneration. The contribution of genetics to the initiation and progression of DR has been recognized for some time, but the involvement of specific genes and genetic variants remains elusive. Several investigations are currently underway for identifying DR susceptibility loci through linkage studies, candidate gene approaches, and genome-wide association studies. Advent of next generation sequencing and high throughput genomic technologies, development of novel bioinformatics tools and collaborations among research teams should facilitate such investigations. Here, we review the current state of genetic studies in DR and discuss reported findings in the context of biochemical, cell biological and therapeutic advances. We propose the development of a consortium in India for genetic studies with large cohorts of patients and controls from limited geographical areas to stratify the impact of the environment. Uniform guidelines should be established for clinical phenotyping and data collection. These studies would permit identification of genetic loci for DR susceptibility in the Indian population and should be valuable for better diagnosis and prognosis, and for clinical management of this blinding disease.

Differential Expression Patterns of Eph Receptors and Ephrin Ligands in Human Cancers

Eph receptors constitute the largest family of receptor tyrosine kinases, which are activated by ephrin ligands that either are anchored to the membrane or contain a transmembrane domain. These molecules play important roles in the development of multicellular organisms, and the physiological functions of these receptor-ligand pairs have been extensively documented in axon guidance, neuronal development, vascular patterning, and inflammation during tissue injury. The recognition that aberrant regulation and expression of these molecules lead to alterations in proliferative, migratory, and invasive potential of a variety of human cancers has made them potential targets for cancer therapeutics. We present here the involvement of Eph receptors and ephrin ligands in lung carcinoma, breast carcinoma, prostate carcinoma, colorectal carcinoma, glioblastoma, and medulloblastoma. The aberrations in their abundances are described in the context of multiple signaling pathways, and differential expression is suggested as the mechanism underlying tumorigenesis.

Role of SV40 Integration Site at Chromosomal Interval 1q21.1 in Immortalized CRL2504 Cells

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by beta-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies.

Novel spliced variants of ionotropic glutamate receptor GluR6 in normal human fibroblast and brain cells are transcribed by tissue specific promoters

The members of the ionotropic glutamate receptor family, namely, a-amino-3-hydroxy-S-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors, are important mediators of the rapid synaptic transmission in the central nervous system. We have investigated the splicing pattern and expression of the kainate receptor subunit GluR6 in human fibroblast cell lines and brain tissue. We demonstrate the expression of GluR6A variant specifically in brain, and four variants, namely, GluR6B, GluR6C, GluR6D and GluR6E in fibroblast cell lines. The variants GluR6D and GluR6E have not been described before, and appear to be specific for non-neuronal cells. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter P(N) and non-neuronal promoter P(NN). We have used RNA ligase mediated RACE and in silico analyses to locate two sets of transcription start sites, and confirmed specific transcripts initiated by P(N) and P(NN) in brain cells and fibroblasts, respectively. The domain structure of variants GluR6D and GluR6E revealed the absence of three transmembrane domains. The lack of these domains suggests that the mature receptors arising from these variant subunits may not function as active channels. Based on these structural features in GluR6D and GluR6E, and the observations that GluR6B, GluR6C, GluR6D and GluR6E are exclusively expressed in non-neuronal cells, it is likely that these receptor subunits function as non-channel signaling proteins.