Controls of cell replication in human lung cancer

Abstract

Identification of signal transduction pathways that regulate cell replication will be critical for understanding the basis of inappropriately controlled replication of neoplastic cells and for the development of strategies to control neoplastic cell replication. The studies described in this dissertation have focused on the regulation of cell replication in a human lung carcinoma cell line EKVX, which appears to be a prototype for human non-small cell lung cancer. The principle strategy used in these studies was pharmacological manipulation of cell replication by protein kinase inhibitors. Replication of these cells, as measured by DNA synthesis and cell growth assays, was inhibited by agents such as staurosporine that have relative specificity for protein kinase C. Treatment of cells with staurosporine was demonstrated to cause effective and reversible arrest of cells in G1 phase of cell cycle (as detected by flow cytometric analysis), which was accompanied by disappearance of the phosphorylated form of retinoblastoma (RB) protein (the inactive form of RB protein) as revealed by Western analysis. Possible involvement of expression of certain oncogenes was also investigated using competitive reverse-transcription polymerase chain reaction (RT-PCR), but no significant changes in c-myc and c-fos expression were observed to correlate with the inhibitory effects of staurosporine. These results suggest that regulation of cell replication in EKVX may be at least in part dependent on the activity of RB protein, which in turn is inactivated (i.e., phosphorylated) by protein kinase(s) sensitive to staurosporine. The activities of c-myc and c-fos may not be important for replication of EKVX. Future studies should address the mechanism employed by RB protein in regulating EKVX replication, the roles of other cellular events which have replication stimulating effects, and differences in regulation of cell replication between normal and neoplastic cells.