CLUSTER-10 LUNG-CANCER ANTIBODIES RECOGNIZE NSPS, NOVEL NEUROENDOCRINE PROTEINS ASSOCIATED WITH MEMBRANES OF THE ENDOPLASMIC-RETICULUM

We have identified a novel gene (the NSP gene) encoding 3 transcripts and coding for 3 neuroendocrine-specific proteins (NSPs), by screening a cDNA expression library of the small-cell lung-cancer (SCLC) cell l ine NC1-H82 with the cluster-10 lung-cancer antibodies RNL2 and RNL3. The 3 transcripts code for NSPs with apparent molecular weights of 135 kDa (NSP-A), 43 to 45 and 35 kDa (NSP-B) and 23 kDa (NSP-C). NSP-A an d NSP-B are recognized by antibodies RNL2 and RNL3, while second-gener ation antibodies, specifically recognizing NSP-A and NSP-C, have been produced after immunization with a hybrid protein obtained after bacte rial expression of the largest NSP-transcript or with a synthetic pept ide specific for NSP-C. The NSPs exhibit a highly restricted distribut ion pattern and are found mainly in neural and neuro-endocrine cell ty pes, and in neuro-endocrine tumours. Of the different types of lung tu mours, mainly SCLC and carcinoids were positive in immunocytochemical assays using the anti-NSP antibodies, while non-SCLC were in general n egative. The subcellular distribution of the NSPs was studied in human SCLC cell lines. They do not co-localize with components typical of n euro-endocrine granules, such as synaptophysin and chromogranin. The u se of NSP antibodies in the immunofluorescence technique applied to cu ltured SCLC cells, made it obvious that these proteins localize in the endoplasmic reticulum. Cell fractionation procedures, monitored by im munoblotting assays, indicated an association of the NSPs with the mic rosomal fraction, from which they could be solubilized with Triton X-1 00. Gel filtration studies with this solubilized fraction revealed tha t NSPs form supramolecular aggregates with a molecular weight of more then 500 kDa. (C) 1994 Wiley-Liss, Inc.