metallothionein and Retinoblastoma

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.

Topics: Animals; Antineoplastic Agents; Carboplatin; Cell Line, Tumor; Cisplatin; Humans; Metallothionein; Models, Biological; Neuroblastoma; Rabbits; Retinoblastoma

Protein kinase C and its family of multiple subspecies play pivotal roles in cell-surface mediated signal transduction. For example, in the process of synthesizing melatonin, the activation of alpha 1-adrenergic receptor sites in the pineal gland causes translocation of protein kinase C, which in turn enhances the beta-adrenergic-activated accumulation of both cyclic AMP and cyclic GMP. In the retina, protein kinase C phosphorylates rhodopsin and hence is involved in visual transduction. The activation of protein kinase C depends on the presence of phospholipid and Ca++. In this communication, we report that the bovine pineal gland and retina possess unique protein kinase C isoenzymes that are distinct from those seen in the rat brain. Furthermore, in retinoblastoma cells in culture, protein kinase C is stimulated by a cooperative interaction between calcium and zinc. Moreover, the subcellular regions of retina that exhibit the highest activity of protein kinase C also possess the highest concentration of zinc. In view of the fact that the bovine pineal gland and retina continually synthesize metallothionein and other low molecular weight zinc binding proteins, we propose that zinc and metallothionein participate in signal transduction in the retina and pineal gland. The action of metallothionein, a zinc binding protein, in activating protein kinase C is opposite to that of calcium binding protein, which is a potent inhibitor of protein kinase C.

Topics: Animals; Brain; Calcium; Cattle; Enzyme Activation; Eye Neoplasms; Isoenzymes; Male; Metallothionein; Pineal Gland; Protein Kinase C; Rats; Rats, Inbred Strains; Retina; Retinoblastoma; Signal Transduction; Subcellular Fractions; Tumor Cells, Cultured; Zinc

In addition to retinoblastoma and osteosarcoma, mutation of both alleles of the RB1 gene occurs frequently in several other types of tumors. In order to evaluate the role of RB1 in cancer, the wild type RB1 gene was introduced into the RB1-deleted breast cancer cell line MDA-468-S4 and retinoblastoma cell lines WERI-Rb1 and Y-79. The RB1 complementary DNA was under control of the inducible murine metallothionein promoter in MDA-468-S4 and the thymidine kinase promoter in the retinoblastoma lines. The protein, p110RB1, produced from the exogenously introduced gene appeared normal by immunoprecipitation, Western blot analysis, and nuclear localization and also showed normal cell cycle-dependent phosphorylation and an ability to bind to E1a protein. No changes in growth rate or morphology were observed in either of the reconstituted cell types. Expression of p110RB1 in MDA-468-S4 did not affect anchorage-independent growth when measured by colony formation in soft agar. Although the ability of WERI-Rb1 cells expressing p110RB1 to form colonies in methylcellulose was reduced, the reconstituted retinoblastoma cell lines formed intraocular tumors in immunodeficient mice with the same efficiency as the RB1-negative parent cell lines and the tumors produced by the RB1-reconstituted cells continued to express p110RB1. These experimental results suggest that the malignant phenotype is little affected by the replacement of p110RB1 and that RB1 is a relatively weak tumor suppressor gene.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line; Chromosome Deletion; Eye Neoplasms; Female; Genes, Retinoblastoma; HeLa Cells; Humans; Metallothionein; Phenotype; Plasmids; Promoter Regions, Genetic; Recombinant Fusion Proteins; Restriction Mapping; Retinoblastoma; Thymidine Kinase; Transfection