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C-terminal flanking peptide of human progastrin is expressed in normal human gastroduodenal mucosa, antra with G cell hyperplasia and islet cell tumours producing gastrin
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EXPRESSION OF C-FLANKING PEPTIOE OF HUMANPRO-BOMBESIN IN PULMONARY AND EXTRA-PULMONARY S~LL CELL CARCINOMA. Q. HAMID, D.R. SPRINGALL, M.A. GHATEI1, B.A. FOUNTAINI , B. ADDIS2, B.N. IBRAHIM3, S.R. BLOOMI & J.M. POLAK. DEPARTMENTSOF HISTOCHEMISTRY AND IMEDICINE, ROYALPOSTGRADUATE MEDICAL SCHOOL. LONDON W12 OHS; DEPARTMENTS OF HISTOPATHOLOGY, 2BROMPTON HOSPITAL, LONDONSW7, AND 3FRENCHAY HOSPITAL, BRISTOL BS16 1LE, U.K. Human-bombesin immunoreactivity is widely distributed throughout the nervous system and the gastrointestinal and respiratory tracts. In the l a t t e r , i t is present in mucosal endocrine cells which are most prevalent in the neonatal period. Bombesinimmunoreactivity is also found in some neuroendocrine tumours, where i t appears to be a significant autocrine growth factor. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro-bombesin. From this the amino acid sequence of the molecule has been predicted and shows the peptide to be encoded in a typical polypeptide precursor consisting of a signal peptide, bombesin i t s e l f , and a carboxy-terminal (C-flanking) extension peptide, the function of which is not yet known. The sequence of the C-terminal of the C-flanking peptide has been shown to be variable; three separate mRNAs have been isolated, representing different transcriptional products from a single gene. We have raised a rabbit antiserum to the predicted N-terminal ( i . e . the conserved region) 30 amino acid sequence of the C-flanking peptide. Surgically resected cases of small cell carcinomas of the lung (n=100) and extra-pulmonary sites (n=28) were evaluated for the presence of both bombesin and the C-flanking peptide of hunkln pro-bombesin using immunocytochemistry and radioimmunoassay. Sections (5 ~m thick) of formalin-fixed wax-embeddedtissue were immunostained by the PAP method. Although only 28 of the pulmonary and 1 of the extra-pulmonary cases showed bombesin immunoreactivity, 65 cases of small cell carcinoma of the lung and 14 cases of extra-pulmonary small cell carcinoma were immunostained by antiserum to the C-flanking peptide. Immunoreactivity was abolished by absorption of each antibody with i t homologous antigen. Radioimmunoassayof extracts of tumour tissue recovered from wax blocks demonstrated C-flanking peptide-like immunoreactivity (241~166 pmol/g of tissue) in small cell carcinoma of the lung (n=11). On SephadexG-50 chromatography the immunoreactive material eluted with a Kav of 0.15. The apparent presence of C-flanking peptide at'higher levels than bombesin in SCC suggests that abnormal mechanismof synthesis, storage or release of the peptide may occur in the tumour c e l l s . Antisera to this C-flanking peptide may therefore prove to be of value in investigating the biological behaviour of small cell carcinomas.
C-TERMINAL FLANKING'PEPTIDE OF HUMAN PROGASTRIN IS EXPRESSED IN NORMALHUMANGASTRODUODENAL MUCOSA, ANTRAWITH G CELL HYPERPLASIA AND ISLET CELL TUMOURS PRODUCING GASTRIN M. HARA, I.M. VARNDELL, A.E~ BISHOP, J. RODE1, S.R. BLOOM2 AND J.M. POLAK, DEPARTMENTS OF HISTOCHEMISTRY AND MEDICINE~, RPMS., DU CANE ROAD, LONDONW12 OHS AND THE BLAND-SUTTON INSTITUTE OF PATHOLOGY1, THE MIDDLESEX HOSPITAL MEDICAL SCHOOL, LONDONWIP 7PN, U.K. Regulatory peptides, probably without exception, are derived from larger precursor molecules. Antisera raised to different regions of the whole precursor generally show a commonlocalisation pattern and thus are of limited value, Notable exceptions include the intragranular segregation of pro-glucagon products in pancreatic alpha granules and the intergranular segregation of epitopes in maturing beta granules, Closely related regulatory peptides may also share some structural s i m i l a r i t i e s which make i t d i f f i c u l t to differentiate separate cell types. One such example is the C-terminal octapeptide commonto both gastrin and cholecystokinin (CCK). We have used antisera generated to the C-terminal peptide unique to human pro-gastrin (PSN; peptide Ser-Ala-Glu-Asp-Glu-Asn) to differentiate gastrointestinal gastrin/CCK cell types. Using this antibody, we have localized PSN (human progastrin) immunoreactivity to cells in human gastroduodenal mucosa (n = 5), antra with G cell hyperplasia (n = 10) and i s l e t cell tumours producing gastrin (n = 6). The results obtained were compared with those of antisera to the C-terminal of gastrin and to the N-terminal of G34. In the gastric antrum, many PSN (human progastrin)-immunoreactive cells were found and showed a similar distribution to G34 and C-terminal gastrin-immunoreactive cells. PSN (human progastrin) immunoreactivity was colocalized with gastrin in antral G cells, At the EM level, PSN (human progastrin) immunoreactivity was associated with each granule type in the antral G cells, i . e . both Rlectron-lucent and electron-dense granules. Hyperplastic G cells, in endoscopic biopsies from pernicious anaemia patients, also showed PSN (human progastrin) immunoreactivity. In the duodenum, PSN (human progastHn)- and G34-immunoreactivecells were scattered, in contrast to the large number of the cells detected by a C-terminal-reacting gastrin antiserum. In the pancreatic tumours, the antisera to PSN, G34 and C-terminal gastrin revealed a similar, variable pattern of localization. None of the cells immunostainedwith the anti-PSN (human progastrin) sera exhibited CCK cell morphology. Thus, we report the existence of C-terminal-flanking peptide of human progastrin in G cells in normal gastro-duodenal mucosa, G cell hyperplasia and i s l e t cell tumours producing gastrin.
EXPRESSION OF C-FLANKING PEPTIOE OF HUMANPRO-BOMBESIN IN PULMONARY AND EXTRA-PULMONARY S~LL CELL CARCINOMA. Q. HAMID, D.R. SPRINGALL, M.A. GHATEI1, B.A. FOUNTAINI , B. ADDIS2, B.N. IBRAHIM3, S.R. BLOOMI & J.M. POLAK. DEPARTMENTSOF HISTOCHEMISTRY AND IMEDICINE, ROYALPOSTGRADUATE MEDICAL SCHOOL. LONDON W12 OHS; DEPARTMENTS OF HISTOPATHOLOGY, 2BROMPTON HOSPITAL, LONDONSW7, AND 3FRENCHAY HOSPITAL, BRISTOL BS16 1LE, U.K. Human-bombesin immunoreactivity is widely distributed throughout the nervous system and the gastrointestinal and respiratory tracts. In the l a t t e r , i t is present in mucosal endocrine cells which are most prevalent in the neonatal period. Bombesinimmunoreactivity is also found in some neuroendocrine tumours, where i t appears to be a significant autocrine growth factor. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro-bombesin. From this the amino acid sequence of the molecule has been predicted and shows the peptide to be encoded in a typical polypeptide precursor consisting of a signal peptide, bombesin i t s e l f , and a carboxy-terminal (C-flanking) extension peptide, the function of which is not yet known. The sequence of the C-terminal of the C-flanking peptide has been shown to be variable; three separate mRNAs have been isolated, representing different transcriptional products from a single gene. We have raised a rabbit antiserum to the predicted N-terminal ( i . e . the conserved region) 30 amino acid sequence of the C-flanking peptide. Surgically resected cases of small cell carcinomas of the lung (n=100) and extra-pulmonary sites (n=28) were evaluated for the presence of both bombesin and the C-flanking peptide of hunkln pro-bombesin using immunocytochemistry and radioimmunoassay. Sections (5 ~m thick) of formalin-fixed wax-embeddedtissue were immunostained by the PAP method. Although only 28 of the pulmonary and 1 of the extra-pulmonary cases showed bombesin immunoreactivity, 65 cases of small cell carcinoma of the lung and 14 cases of extra-pulmonary small cell carcinoma were immunostained by antiserum to the C-flanking peptide. Immunoreactivity was abolished by absorption of each antibody with i t homologous antigen. Radioimmunoassayof extracts of tumour tissue recovered from wax blocks demonstrated C-flanking peptide-like immunoreactivity (241~166 pmol/g of tissue) in small cell carcinoma of the lung (n=11). On SephadexG-50 chromatography the immunoreactive material eluted with a Kav of 0.15. The apparent presence of C-flanking peptide at'higher levels than bombesin in SCC suggests that abnormal mechanismof synthesis, storage or release of the peptide may occur in the tumour c e l l s . Antisera to this C-flanking peptide may therefore prove to be of value in investigating the biological behaviour of small cell carcinomas.
C-TERMINAL FLANKING'PEPTIDE OF HUMAN PROGASTRIN IS EXPRESSED IN NORMALHUMANGASTRODUODENAL MUCOSA, ANTRAWITH G CELL HYPERPLASIA AND ISLET CELL TUMOURS PRODUCING GASTRIN M. HARA, I.M. VARNDELL, A.E~ BISHOP, J. RODE1, S.R. BLOOM2 AND J.M. POLAK, DEPARTMENTS OF HISTOCHEMISTRY AND MEDICINE~, RPMS., DU CANE ROAD, LONDONW12 OHS AND THE BLAND-SUTTON INSTITUTE OF PATHOLOGY1, THE MIDDLESEX HOSPITAL MEDICAL SCHOOL, LONDONWIP 7PN, U.K. Regulatory peptides, probably without exception, are derived from larger precursor molecules. Antisera raised to different regions of the whole precursor generally show a commonlocalisation pattern and thus are of limited value, Notable exceptions include the intragranular segregation of pro-glucagon products in pancreatic alpha granules and the intergranular segregation of epitopes in maturing beta granules, Closely related regulatory peptides may also share some structural s i m i l a r i t i e s which make i t d i f f i c u l t to differentiate separate cell types. One such example is the C-terminal octapeptide commonto both gastrin and cholecystokinin (CCK). We have used antisera generated to the C-terminal peptide unique to human pro-gastrin (PSN; peptide Ser-Ala-Glu-Asp-Glu-Asn) to differentiate gastrointestinal gastrin/CCK cell types. Using this antibody, we have localized PSN (human progastrin) immunoreactivity to cells in human gastroduodenal mucosa (n = 5), antra with G cell hyperplasia (n = 10) and i s l e t cell tumours producing gastrin (n = 6). The results obtained were compared with those of antisera to the C-terminal of gastrin and to the N-terminal of G34. In the gastric antrum, many PSN (human progastrin)-immunoreactive cells were found and showed a similar distribution to G34 and C-terminal gastrin-immunoreactive cells. PSN (human progastrin) immunoreactivity was colocalized with gastrin in antral G cells, At the EM level, PSN (human progastrin) immunoreactivity was associated with each granule type in the antral G cells, i . e . both Rlectron-lucent and electron-dense granules. Hyperplastic G cells, in endoscopic biopsies from pernicious anaemia patients, also showed PSN (human progastrin) immunoreactivity. In the duodenum, PSN (human progastHn)- and G34-immunoreactivecells were scattered, in contrast to the large number of the cells detected by a C-terminal-reacting gastrin antiserum. In the pancreatic tumours, the antisera to PSN, G34 and C-terminal gastrin revealed a similar, variable pattern of localization. None of the cells immunostainedwith the anti-PSN (human progastrin) sera exhibited CCK cell morphology. Thus, we report the existence of C-terminal-flanking peptide of human progastrin in G cells in normal gastro-duodenal mucosa, G cell hyperplasia and i s l e t cell tumours producing gastrin.