Cluster-10 lung-cancer antibodies recognize NSPs, novel neuro-endocrine 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 line NCI-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 and NSP-B are recognized by antibodies RNL2 and RNL3, while second-generation antibodies, specifically recognizing NSP-A and NSP-C, have been produced after immunization with a hybrid protein obtained after bacterial expression of the largest NSP-transcript or with a synthetic peptide specific for NSP-C. The NSPs exhibit a highly restricted distribution pattern and are found mainly in neural and neuro-endocrine cell types, and in neuro-endocrine tumours. Of the different types of lung tumours, mainly SCLC and carcinoids were positive in immunocytochemical assays using the anti-NSP antibodies, while non-SCLC were in general negative. The subcellular distribution of the NSPs was studied in human SCLC cell lines. They do not co-localize with components typical of neuro-endocrine granules, such as synaptophysin and chromogranin. The use of NSP antibodies in the immunofluorescence technique applied to cultured SCLC cells, made it obvious that these proteins localize in the endoplasmic reticulum. Cell fractionation procedures, monitored by immunoblotting assays, indicated an association of the NSPs with the microsomal fraction, from which they could be solubilized with Triton X-100. Gel filtration studies with this solubilized fraction revealed that NSPs form supramolecular aggregates with a molecular weight of more then 500 kDa.
Senden
N H
NH
Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, The Netherlands.
van de Velde
H J
HJ
Broers
J L
JL
Timmer
E D
ED
Roebroek
A J
AJ
van de Ven
W J
WJ
Ramaekers
F C
FC
D000818:Animals; D000912:Antibodies, Neoplasm / Q000276:immunology*; D018288:Carcinoma, Small Cell / Q000276:immunology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D008175:Lung Neoplasms / Q000276:immunology*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D016366:Open Reading Frames; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D000939:Epitopes; D009419:Nerve Tissue Proteins; C081781:RTN1 protein, human