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Dynamic proliferation assessment of lung cancer with in vivo BrdU labeling and bivariate DNA flowcytometry
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Dynamic proliferation assessment of lung cancer with in viva BrdU labeling and bivariate DNA flowcytometry. G. ten Velde, M. Tinnemans, M. Lenders, B. Schutte, G. Blijham. Department of Pulmonology, Internal Medicine and Molecular Celbiology, University of Limburg, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. In several human malignancies including lung cancer, proliferative characteristics such as the percentage of S-phase cells (SPF) in DNA flowcytometry (FCM) and ex viva tritiated thymidine labeling index (LI) have been found to be prognostically and possibly therapeutically relevant. These assays however are sensitive to methodological errors and do not reflect dynamic parameters such as the S-phase transit time (Ts) and potential tumor doubling time (Tpot). We took bronchoscopic biopsies 4 hours after in viva labeling with the DNA-precursor Bromodeoxyuridine (BrdU). Single nuclear suspensions of these biopsies were stained with propidium iodide (for DNA) and anti-BrdU monoclonal antibody and subsequently analysed by bivariate FCM. To date, 59 patients have been labeled without side effects. Full data are available for 24 patients, of whom 9 had small cell lung cancer and 46% were aneuploid. The following cytokinetic data were obtained (mean and SD): LI 9% (8.5), Ts 9.1 hour (3.91, Tpot 264 hours (318). Analysis of a larger number of patients and correlations with histology and ploidy level will be presented. In conclusion, in viva labeling of bronchoscopic specimens with BrdU is a feasible and safe procedure to obtain dynamic cytokinetic information of lung cancer.
A McAb LC-1 WHICH AGAINST HUMAN LUNG CANCER X.R. Ge, J. Wang, S.J. Lin, Y.F. Che and M. Li (Shanghai Institute of Cell Biology, CAS) Recently, Ge et al reported LAC McAbs against human non-small cell lung cancer and LSC McAbs against human small cell carcinoma. Only few reports deal with the common epitopes of the lung cancers. One McAb from 20 hybrids from a fusion, named LC-1,has been obtained. The results of ABC staining of LC-1 with a variety of tumors, in 48 human lung cancers, 21/22 adenocarcinoma, lo/11 squamacarcinoma, 3/3 large cell carcinoma, 5/5 mixed tumor and 7/7 small cell carcimoma showed the positive staining. Apart from l/4 normal human kidney, all the other 15 normal human tissues and 12 sixth months foetal tissues showed negative reaction. The RIA test showed the lung adenocarcinoma antigen associated binding sites with'? LC-1 was about 7.2X104sited per cell. The affinity constant of LC-1 was 4.8X10 M:8 3 bands, M.W. are 91K, 70K and 51K, could be seen in immunoblot of LC-1 reacted with the extracts of lung adenocarcinoma cells. They are all reacted with staining of alcian blue but only two bands, 70K and 51K, staining by Sudan black. When cells ti-eacted with proteinase K and sodium period&e separately, and then performed RIA and FACS, sodium periodate treatment could influence McAb LC-1 binding to target cells mole significantly than proteinase k treated cells did. It was shown that the tumor associated antigens of LC-1 against was glycoproteins in adenocarcinoma and the epitope may probably be polysacchrides.
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IMMUNOHISTOCHEMICAL LOCALIZATION OF PARATHYROID HORMONE-RELATED PROTEIN IN BRONCHIAL CARCINOMA. J.A. Hayman*, J. Southby, J.A. Danks, J.M. Moseley, T.J. *Dept. of Pathology, University of Melbourne, Martin. Parkville 3052 and St. Vincent’s Institute of Medical Research, Fitzroy 3065, Australia.
ROLE OF PROTEOLYTICSYSTRM IN LUNG MR'USTASESDEVEUXNEXTAT EXPERIMCNTALTGMORSINMICE. S.G.CRUBINSKAU,V.B.VINNITSKY.Institutefor Oncology and RadiobiologY Problems Aead.Sci. of Ukranian S.S.Ft.,Kiev, USSR The activityof elastase,collagenase,dl-protease inhibitorand d 2-maoroglobulin have been studied in the mice with metastatic(Lewis Lung caroinoma,Melanoma ~-16) and non-metastatio (Mamtmry gland adenooarcinona 755) tumors. Primary tumors and lung tissues (organ of metastatiolesion)have been studied in growth dynamicsand tumor metastatiospreading.One could observethe increasedactivitiesof elastaseand oollagenasein mice with metastatictumors both in primarytumor tissues and lungs. The maximal increase of elastaseactivity in tumor and lung tissueshas been observedduring the period of metastasesstroma formationand maximal increaseof collagenaseactivity at the time of metastaticgrowth. Lewis lung carcinoma and melanoma~-16 have shown a low level ofdl-protease inhibitorand d2-maoroglobuli.n activities. The activitiesof these inhibitorsin Mannnary gland adenocarcinoma755 were reliablyhigher than in metastatic tumors. Tcielin,the specificelastaseinhibitor,used sepurutelyand combinedwith oyolophosphamide suppressed elastaseactivitiesin tumors and lungs and inhibited metastasesdevelopent in lungs. There was drawn the conclusionof the importantrole of elastase,collagenaseand their inhibitorsin lung metsstasesdevelopnent.
Parathyroid hormone related protein (PTHrP) is a major contributing factor in the aetiology of humeral hypemalcaemia of malignancy (HHM). HHM is often a complication of squamous cell carcinoma of the bronchus but may occur with tumours of Hypercalcaemia occurs in 10% of other epithelial surfaces. patients with advanced bronchial cancer and a bronchial squamous carcinoma cell line has beena source ofPTHrP. Polyclonal antibodies have been raised in rabbits against synthetic (l-34) peptides. This antiserum is highly specific and does not cross-react with PTH on Western blot, in radioimmunoassay or at high concentrations to block adenylate Using this antiserum and cyclase in PTH responsive cells. peroxidase-antiperoxidase procedures PTHrP has been identified in the cells of squamous cell carcinoma of the bronchus and in bronchial epithelium showing squamous metaplasia. PTHrP staining is absent from normal brochial epithelium but is present uniformly in squamous cell carcinomas in patients with and without hypemalcaemia. Demonstration of the PlHrP antigen in malignant bronchial tumour tissue provides an explanation for the association of HHM with these tumours. It is possible that the hormone may also be at least in part responsible for the ability of these tumours to absorb and to metastasize to bone.
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Dynamic proliferation assessment of lung cancer with in viva BrdU labeling and bivariate DNA flowcytometry. G. ten Velde, M. Tinnemans, M. Lenders, B. Schutte, G. Blijham. Department of Pulmonology, Internal Medicine and Molecular Celbiology, University of Limburg, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. In several human malignancies including lung cancer, proliferative characteristics such as the percentage of S-phase cells (SPF) in DNA flowcytometry (FCM) and ex viva tritiated thymidine labeling index (LI) have been found to be prognostically and possibly therapeutically relevant. These assays however are sensitive to methodological errors and do not reflect dynamic parameters such as the S-phase transit time (Ts) and potential tumor doubling time (Tpot). We took bronchoscopic biopsies 4 hours after in viva labeling with the DNA-precursor Bromodeoxyuridine (BrdU). Single nuclear suspensions of these biopsies were stained with propidium iodide (for DNA) and anti-BrdU monoclonal antibody and subsequently analysed by bivariate FCM. To date, 59 patients have been labeled without side effects. Full data are available for 24 patients, of whom 9 had small cell lung cancer and 46% were aneuploid. The following cytokinetic data were obtained (mean and SD): LI 9% (8.5), Ts 9.1 hour (3.91, Tpot 264 hours (318). Analysis of a larger number of patients and correlations with histology and ploidy level will be presented. In conclusion, in viva labeling of bronchoscopic specimens with BrdU is a feasible and safe procedure to obtain dynamic cytokinetic information of lung cancer.
A McAb LC-1 WHICH AGAINST HUMAN LUNG CANCER X.R. Ge, J. Wang, S.J. Lin, Y.F. Che and M. Li (Shanghai Institute of Cell Biology, CAS) Recently, Ge et al reported LAC McAbs against human non-small cell lung cancer and LSC McAbs against human small cell carcinoma. Only few reports deal with the common epitopes of the lung cancers. One McAb from 20 hybrids from a fusion, named LC-1,has been obtained. The results of ABC staining of LC-1 with a variety of tumors, in 48 human lung cancers, 21/22 adenocarcinoma, lo/11 squamacarcinoma, 3/3 large cell carcinoma, 5/5 mixed tumor and 7/7 small cell carcimoma showed the positive staining. Apart from l/4 normal human kidney, all the other 15 normal human tissues and 12 sixth months foetal tissues showed negative reaction. The RIA test showed the lung adenocarcinoma antigen associated binding sites with'? LC-1 was about 7.2X104sited per cell. The affinity constant of LC-1 was 4.8X10 M:8 3 bands, M.W. are 91K, 70K and 51K, could be seen in immunoblot of LC-1 reacted with the extracts of lung adenocarcinoma cells. They are all reacted with staining of alcian blue but only two bands, 70K and 51K, staining by Sudan black. When cells ti-eacted with proteinase K and sodium period&e separately, and then performed RIA and FACS, sodium periodate treatment could influence McAb LC-1 binding to target cells mole significantly than proteinase k treated cells did. It was shown that the tumor associated antigens of LC-1 against was glycoproteins in adenocarcinoma and the epitope may probably be polysacchrides.
117
118
IMMUNOHISTOCHEMICAL LOCALIZATION OF PARATHYROID HORMONE-RELATED PROTEIN IN BRONCHIAL CARCINOMA. J.A. Hayman*, J. Southby, J.A. Danks, J.M. Moseley, T.J. *Dept. of Pathology, University of Melbourne, Martin. Parkville 3052 and St. Vincent’s Institute of Medical Research, Fitzroy 3065, Australia.
ROLE OF PROTEOLYTICSYSTRM IN LUNG MR'USTASESDEVEUXNEXTAT EXPERIMCNTALTGMORSINMICE. S.G.CRUBINSKAU,V.B.VINNITSKY.Institutefor Oncology and RadiobiologY Problems Aead.Sci. of Ukranian S.S.Ft.,Kiev, USSR The activityof elastase,collagenase,dl-protease inhibitorand d 2-maoroglobulin have been studied in the mice with metastatic(Lewis Lung caroinoma,Melanoma ~-16) and non-metastatio (Mamtmry gland adenooarcinona 755) tumors. Primary tumors and lung tissues (organ of metastatiolesion)have been studied in growth dynamicsand tumor metastatiospreading.One could observethe increasedactivitiesof elastaseand oollagenasein mice with metastatictumors both in primarytumor tissues and lungs. The maximal increase of elastaseactivity in tumor and lung tissueshas been observedduring the period of metastasesstroma formationand maximal increaseof collagenaseactivity at the time of metastaticgrowth. Lewis lung carcinoma and melanoma~-16 have shown a low level ofdl-protease inhibitorand d2-maoroglobuli.n activities. The activitiesof these inhibitorsin Mannnary gland adenocarcinoma755 were reliablyhigher than in metastatic tumors. Tcielin,the specificelastaseinhibitor,used sepurutelyand combinedwith oyolophosphamide suppressed elastaseactivitiesin tumors and lungs and inhibited metastasesdevelopent in lungs. There was drawn the conclusionof the importantrole of elastase,collagenaseand their inhibitorsin lung metsstasesdevelopnent.
Parathyroid hormone related protein (PTHrP) is a major contributing factor in the aetiology of humeral hypemalcaemia of malignancy (HHM). HHM is often a complication of squamous cell carcinoma of the bronchus but may occur with tumours of Hypercalcaemia occurs in 10% of other epithelial surfaces. patients with advanced bronchial cancer and a bronchial squamous carcinoma cell line has beena source ofPTHrP. Polyclonal antibodies have been raised in rabbits against synthetic (l-34) peptides. This antiserum is highly specific and does not cross-react with PTH on Western blot, in radioimmunoassay or at high concentrations to block adenylate Using this antiserum and cyclase in PTH responsive cells. peroxidase-antiperoxidase procedures PTHrP has been identified in the cells of squamous cell carcinoma of the bronchus and in bronchial epithelium showing squamous metaplasia. PTHrP staining is absent from normal brochial epithelium but is present uniformly in squamous cell carcinomas in patients with and without hypemalcaemia. Demonstration of the PlHrP antigen in malignant bronchial tumour tissue provides an explanation for the association of HHM with these tumours. It is possible that the hormone may also be at least in part responsible for the ability of these tumours to absorb and to metastasize to bone.