The overall objective of this research proposal is to gain a detailed understanding of the molecular and cellular mechanisms underlying the invasion and metastasis of human small cell lung carcinoma (SCCL). This objective will be approached by conducting in depth studies into the structure and expression of two, distinct glycoprotein antigens that are preferentially expressed by SCCL cells. The rationale underlying this proposal is to initiate these molecular analyses by capitalizing on monoclonal antibodies that recognize a 100 kDa glycoprotein secreted by SCCL and by other neuroectodermal tumors, and a sulfated, 90 kDa cell surface phospho-glycoprotein strongly associated with SCCL that may lead to a better understanding of the mechanisms of neoplastic transformation. The monoclonal antibodies that have faciliated the preliminary characterization of these molecules will now serve as tools in their purification from extracts and spent culture medium of SCCL and melanoma cells. Specifically, after isolating and deglycosylating each SCCL- associated molecule, high affinity polyclonal antisera will be produced against the protein backbone that will be used to screen phage expression libraries constructed form SCCL mRNA. In order to isolate full length cDNAs corresponding to each of the two SCCL target antigens, preliminary clones isolated by immunostaining of phage plaques will be used to screen an Okayama-Berg library by nucleic acid hybridization. From the cDNA clones, RNA will be transcribed in vitro, by way of transcription vector system, that will lead to the respective complete nucleic acid coding sequences and deduced protein sequences. Using the in vitro transcribed RNA probes, the expression of these two antigens will be studied in detail at the tissue level by in situ hybridization. It is anticipated that the approaches outline in this proposal will lead to identification of two distinct SCCL-associated molecules and possible to a better understanding of their role in tumor biology.
NIH ExPorter
NIH ExPorter