calpain and Retinoblastoma

We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments.. CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions.. Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains.. CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Topics: Animals; Blotting, Western; Calpain; Cattle; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Mice; Neoplasms, Experimental; Photoreceptor Cells; Retina; Retinal Neoplasms; Retinoblastoma; RNA, Neoplasm; Synapses; Tumor Cells, Cultured

Retinoblastoma (Rb) is the most common intraocular malignancy of childhood. Although systemic and intrathecal chemotherapy with local and cranial radiotherapy have improved overall survival, the prognosis for patients with central nervous system involvement is still poor. We investigated the role of the transcription factor nuclear factor (NF)-kappaB, which promotes cell survival in several other models, in the pathophysiology of Rb. The human Rb cell lines Y79 and WERI-Rb1 were treated with the cell permeable peptide SN50, that specifically inhibits the transcriptional activity of NF-kappaB by blocking its translocation into the nucleus. We found that NF-kappaB inhibition up-regulated Bax; down-regulated the anti-apoptotic proteins Bcl-2, A1, and cIAP-2; and induced loss of the mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis in Rb cells. Inhibition of the p38 kinase sensitized cells to SN50-induced cell death, whereas insulin-like growth factor-1 activated NF-kappaB and attenuated the proapoptotic effect of SN50. Finally, NF-kappaB inhibition sensitized Rb cells to doxorubicin. In conclusion, inhibition of NF-kappaB activity in Rb cells leads to loss of mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis. Therapeutic strategies targeting NF-kappaB could be beneficial in the clinical management of Rb, either alone or in combination with conventional chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Calpain; Caspase 3; Caspases; Cell Survival; Dipeptides; DNA; Doxorubicin; Enzyme Inhibitors; Humans; Imidazoles; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Oligonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Peptides; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma; Tumor Cells, Cultured