The regulation of cell cycle progression is disrupted in neoplastically transformed cells, often because proteins controlling the cell cycle are abnormally expressed or activated. The objective of this proposal is to identify proteins which control cell cycle progression in small cell lung carcinoma (SCLC), a highly metastatic tumor which causes over 25,000 deaths per year in the United States. A unique approach will be used to identify these proteins, based on my finding that activation of M3 muscarinic acetylcholine receptors (mAChR) inhibits cell cycle progression in SCLC cells. Cell cycle regulatory proteins will be distinguished by two criteria. First, the proteins must be modified (by changes in transcription, translation, or posttranslational processing) when SCLC cells progress through the cell cycle. Second, these modifications must not occur when cell cycle progression is inhibited by mAChR activation. This novel approach may also identify oncogenic proteins in SCLC cells. mAChR stimulation may uniquely inhibit SCLC proliferation because it normalizes the aberrant expression or activation of proteins contributing to SCLC transformation. For example, mAChR stimulation may induce the expression or activation of tumor suppressor proteins that are underexpressed or inactivated in SCLC cells. Characterization of these affected proteins may shed light on processes contributing to SCLC transformation. Proteins undergoing cell cycle-dependent, post-transcriptional modifications that are inhibited by mAChR activation will be detected by their unique 35S- or 32P-radiolabelling in cycling SCLC cells, compared to cells that are quiescent or treated with carbachol (an mAChR agonist). These proteins will be identified using antibodies directed against known regulatory proteins and by protein micro-sequence analysis. Northern blot and nuclear run-off transcription assays will identify mRNA transcripts whose cell cycle-dependent accumulation is altered by mAChR activation. To expedite the search for regulatory proteins, southern blot analysis will be used to identify proteins unable to participate in mAChR-mediated growth inhibition because of genetic deletion or rearrangement. Once potential regulatory proteins are identified, antibody and oligonucleotide probes will be made in order to compare the expression and activity of the identified proteins in SCLC and other tissues. Unique expression or activation of these proteins in SCLC cells, compared to other cell types, will provide compelling evidence that the identified proteins contribute to neoplastic transformation in SCLC.
