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Effect of hepatocyte growth factor (HGF) on proliferation of human lung cancer cell lines
808
887 EFFECT OF HEPATOCYTE
GROWTH
FACTOR (HGF)
These findings indicate that HGF dose not serve on human lung cancer cells as a stimulator but as an inhibitor of cell proliferation.
EXPRESSION OF GCZl&212L IN SMALL CELL LUNG CANCER (SCLC) CELLS INHIBITS IN VITRO GROWTH. L.E. Heasleyt, F.M. Mitchell*, J. ~amarripal and G.L. Johnson* lUniversity of Colorado Health Sciences Center and the iNational Jewish Center for ItnmunoloevYI and Respiratory Medicine,Denver, CO80262. Autocrine stimulation ofmitogenesis through G proteincoupled neuropeptide receptors is implicated in the transformed growth of SCLC. To define the dominant G protein pathways that mediate signalling through the involved neuropeptide receptors, we have designed retroviruses to overexpress the mutated a subunit polypeptides in SCLC limes. Expression of ai2Q205L or uoQ205L in NCI-H69,H345 or N417 had little or no effect on the growth of these lines in liquid culture or in soft agar. This finding is consistent with our finding that pertussis toxin has little appreciable influence on the growth of SCLC lines. In contrast, Gq polypeptides are predicted to be involved in neuropeptide signalhng in SCLC since PLC activation and Ca*+ mobilization are observed in response to bombesin and vasopressin. When a constitutive active form of an a -lie polypeptide, al6Q212L, was expressed in the SCLC &es, growth of the cells in soft agar was reduced 70 to 90%. The cells expressing CCl6Q212L exhibited elevated basal PLC activity consistent with the ability of the transfected polypeptide to couple to PLCP. However, the ability of bombesin to regulate PLC activity was ablated, suggesting that the cells are now refractory to neurolmptides. Introduction of Ul6Q212L into rat PC12 pheochromocytoma cells, which exhibit neuroendoctine features similar to SCLC, induces neuronal differentiation, indicating that a growth arrested, more differentiated state may also occur in SCLC in response to CZleQ212L eXpESSiOn.
009
010
ERPATOCYTR GROWTH FRCTOR-SCRTTRR FACTOR (RGF/SF)-YET AUTOCRIRR LOOP II ROY-SMALL CELL LURG CRRCIRONR (RSCU:). M.-S. Tsao, H.
CONWITUTIW ACTIVATION OF PROTEIN KINASE C IN A
ON PROLIFERATION OF HUMAN LUNG CANCER CELL LINES. T. Iijima, T. Kamimuro, T. Yazawa, H. Horiguchi, Department of Patholoav, Y. Nakamura, T. Oeata. __ Instituteof Basic Medic@Sciences, University of Tsukuba, Tsukuba, Japan. Hepatocytegrowth factor(HGF) (Nakamuraetal., Nature 342: 440, 1989), a growth factor which extremely stimulates the proliferation of hepatccytes, was recently reported to react as a growth stimulator on various epithelial cells including alveolar pneumocytes. Futhermore, Rygaard et al. (Br. J. Cancer 6 7: 37, 1993) reported the expression of receptors for HGF in human lung cancer cells. It is, however, still unclear whether the growth factor reacts on proliferation of lung cancer cells as a stimulator. The objective of this study is to clarify the effect of HGF on human lung cancer cells. Seveml cancer cell lines originated from human lung cancers of various histologic types (TKB-2,3/l, 4, 12, 15) and NIH 3T3 were cultured with the basic medium containing HGF (1, 10, 100 @ml) and then number of cells were calculated by MTT assay. As a result, HGF did not stimulate the proliferation of any human lung cancer cells. HGF rather suppressed the cell growth in TKB-2,311 and 12, which were originated from small cell carcinoma and large cell carcinoma of human Iunv
Zhu, A. Giaid, J. Viallet. Upts. of pathology and Oncology, Montreal General Hospital and McGill University,Nontreal, Quebec, Canada H3G lA4. HGF/SF is a multifunctionalcytokine with regulatory roles in the growth, morphogenesis, differentiationand carcinogenesisof epithelial cells. The protein product of met proto-oncoqene is the receptor for HGF/SF. We reported that human normal bronchial epithelial (NHE) and NSCLC cell lines eXpreSSed an autocrinelyactive HGF/SF (Cell Growth 6 Diff., 4: 571-579, 1993). Using in-situ hybridizationand immunologicaltechniques, we have demonstrated that NBH and NSCLC cells in vivo also expressed the HGP/SF and met ~RNAand inmrmnoreactive(IR) proteins. Bronchial epithelium expressed high levei of both the HGP/SF-IR and WET-IR proteins. Primary NSCLCs denronstrated either similar or lower-levelsof these proteins. The level of HGF/SF-IR, but not WET-IR was positively correlated with differentiationin both adenocarcinomas(AUC)and squamous cell carcinomas (SQCC). In AX, there was an inversed correlation between both HGF/SF-IR and NET-IR with the size of the primary tumors, while in SGCC, the HGF/SF-IR but not RRT-IR showed a tendency for positive correlation with the tumor size. Both NRT-IR and HGF/SF-IR did not correlate with tumor stage or metastasis to the regional lymph nodes. These results suggest that HGF/SF-NRT autocrine loop plays important roles in the growth of NSCLC, and the effect may be dependent on the type or differentiationof these tumors.
HUMAN ADENOCARC!INOMA OF TBE LUNG. M. Daya-Makin, F. Wang, Y. Kitagawa, V. Duronio. Pulmonary Rewarch Momtory and The Department of Medicine, University of British Columbia, Vancouver, B.C. Canada V6Z lY6. Protein phoaphorylation, catalyzed by protein kinasea, plays a critical role in the proliferation of normal and cancer cella. We have analyzed Non-Small Cell Lung Cancux (NSCLCs) for conatitutive activation of protein ldnaaes. Tkae atudiea were baaed on the hypothesis that the analysis of protein kinaacs that are cc&tutiveJy activated in the tumors may lead to insights into aigMliq pathways that are abnormally activated in lung cancera. Protein extwta, derived from NSCLCs, were resolved by anion exchange chroma&graphy on a Mono Q column and fractions were assayed h vitro for kiwe activity. By this appmach, we have previously identified a 40 kilodalton tumor-ass&a&d ptutein kinase (designated p4uTAK) that exhibited a higher basal level of activity in adatoearcinomas and squamous cell careinotnas of the lung; in compariaoe to the nonneoplastic lung psnmchyma awounding the tumor. ‘l%e biochemical charac&stics of p4OTAK wae similar to the catalytic subunit of proteinkinasec. Herewereportthatinadditiontop4oTAK,pmteia kinaae C (PKC) iaoform was also conatimtively activated in extracts derived from an -ma of the lung. Immunomadivity to PKC
co-eh~tedwithp4OTAK,byMonoQ chromatography. HewsWar, in conuust tcPKC, the activity of p40TAK was not inbiiited when the kinaaeaaaayawaepafamcdinthepfesauxofapaeudwbatrateof PKC. ‘Ikae studies suggest that p4UTAK is diatiwt from PKC.
ActivationofPKC,inthisadcwcarcinoma,raisestheporsibilitytha PKCdepen&nt signal ba&uction tumors of this histological type.
pathways may play a role in lung
887 EFFECT OF HEPATOCYTE
GROWTH
FACTOR (HGF)
These findings indicate that HGF dose not serve on human lung cancer cells as a stimulator but as an inhibitor of cell proliferation.
EXPRESSION OF GCZl&212L IN SMALL CELL LUNG CANCER (SCLC) CELLS INHIBITS IN VITRO GROWTH. L.E. Heasleyt, F.M. Mitchell*, J. ~amarripal and G.L. Johnson* lUniversity of Colorado Health Sciences Center and the iNational Jewish Center for ItnmunoloevYI and Respiratory Medicine,Denver, CO80262. Autocrine stimulation ofmitogenesis through G proteincoupled neuropeptide receptors is implicated in the transformed growth of SCLC. To define the dominant G protein pathways that mediate signalling through the involved neuropeptide receptors, we have designed retroviruses to overexpress the mutated a subunit polypeptides in SCLC limes. Expression of ai2Q205L or uoQ205L in NCI-H69,H345 or N417 had little or no effect on the growth of these lines in liquid culture or in soft agar. This finding is consistent with our finding that pertussis toxin has little appreciable influence on the growth of SCLC lines. In contrast, Gq polypeptides are predicted to be involved in neuropeptide signalhng in SCLC since PLC activation and Ca*+ mobilization are observed in response to bombesin and vasopressin. When a constitutive active form of an a -lie polypeptide, al6Q212L, was expressed in the SCLC &es, growth of the cells in soft agar was reduced 70 to 90%. The cells expressing CCl6Q212L exhibited elevated basal PLC activity consistent with the ability of the transfected polypeptide to couple to PLCP. However, the ability of bombesin to regulate PLC activity was ablated, suggesting that the cells are now refractory to neurolmptides. Introduction of Ul6Q212L into rat PC12 pheochromocytoma cells, which exhibit neuroendoctine features similar to SCLC, induces neuronal differentiation, indicating that a growth arrested, more differentiated state may also occur in SCLC in response to CZleQ212L eXpESSiOn.
009
010
ERPATOCYTR GROWTH FRCTOR-SCRTTRR FACTOR (RGF/SF)-YET AUTOCRIRR LOOP II ROY-SMALL CELL LURG CRRCIRONR (RSCU:). M.-S. Tsao, H.
CONWITUTIW ACTIVATION OF PROTEIN KINASE C IN A
ON PROLIFERATION OF HUMAN LUNG CANCER CELL LINES. T. Iijima, T. Kamimuro, T. Yazawa, H. Horiguchi, Department of Patholoav, Y. Nakamura, T. Oeata. __ Instituteof Basic Medic@Sciences, University of Tsukuba, Tsukuba, Japan. Hepatocytegrowth factor(HGF) (Nakamuraetal., Nature 342: 440, 1989), a growth factor which extremely stimulates the proliferation of hepatccytes, was recently reported to react as a growth stimulator on various epithelial cells including alveolar pneumocytes. Futhermore, Rygaard et al. (Br. J. Cancer 6 7: 37, 1993) reported the expression of receptors for HGF in human lung cancer cells. It is, however, still unclear whether the growth factor reacts on proliferation of lung cancer cells as a stimulator. The objective of this study is to clarify the effect of HGF on human lung cancer cells. Seveml cancer cell lines originated from human lung cancers of various histologic types (TKB-2,3/l, 4, 12, 15) and NIH 3T3 were cultured with the basic medium containing HGF (1, 10, 100 @ml) and then number of cells were calculated by MTT assay. As a result, HGF did not stimulate the proliferation of any human lung cancer cells. HGF rather suppressed the cell growth in TKB-2,311 and 12, which were originated from small cell carcinoma and large cell carcinoma of human Iunv
Zhu, A. Giaid, J. Viallet. Upts. of pathology and Oncology, Montreal General Hospital and McGill University,Nontreal, Quebec, Canada H3G lA4. HGF/SF is a multifunctionalcytokine with regulatory roles in the growth, morphogenesis, differentiationand carcinogenesisof epithelial cells. The protein product of met proto-oncoqene is the receptor for HGF/SF. We reported that human normal bronchial epithelial (NHE) and NSCLC cell lines eXpreSSed an autocrinelyactive HGF/SF (Cell Growth 6 Diff., 4: 571-579, 1993). Using in-situ hybridizationand immunologicaltechniques, we have demonstrated that NBH and NSCLC cells in vivo also expressed the HGP/SF and met ~RNAand inmrmnoreactive(IR) proteins. Bronchial epithelium expressed high levei of both the HGP/SF-IR and WET-IR proteins. Primary NSCLCs denronstrated either similar or lower-levelsof these proteins. The level of HGF/SF-IR, but not WET-IR was positively correlated with differentiationin both adenocarcinomas(AUC)and squamous cell carcinomas (SQCC). In AX, there was an inversed correlation between both HGF/SF-IR and NET-IR with the size of the primary tumors, while in SGCC, the HGF/SF-IR but not RRT-IR showed a tendency for positive correlation with the tumor size. Both NRT-IR and HGF/SF-IR did not correlate with tumor stage or metastasis to the regional lymph nodes. These results suggest that HGF/SF-NRT autocrine loop plays important roles in the growth of NSCLC, and the effect may be dependent on the type or differentiationof these tumors.
HUMAN ADENOCARC!INOMA OF TBE LUNG. M. Daya-Makin, F. Wang, Y. Kitagawa, V. Duronio. Pulmonary Rewarch Momtory and The Department of Medicine, University of British Columbia, Vancouver, B.C. Canada V6Z lY6. Protein phoaphorylation, catalyzed by protein kinasea, plays a critical role in the proliferation of normal and cancer cella. We have analyzed Non-Small Cell Lung Cancux (NSCLCs) for conatitutive activation of protein ldnaaes. Tkae atudiea were baaed on the hypothesis that the analysis of protein kinaacs that are cc&tutiveJy activated in the tumors may lead to insights into aigMliq pathways that are abnormally activated in lung cancera. Protein extwta, derived from NSCLCs, were resolved by anion exchange chroma&graphy on a Mono Q column and fractions were assayed h vitro for kiwe activity. By this appmach, we have previously identified a 40 kilodalton tumor-ass&a&d ptutein kinase (designated p4uTAK) that exhibited a higher basal level of activity in adatoearcinomas and squamous cell careinotnas of the lung; in compariaoe to the nonneoplastic lung psnmchyma awounding the tumor. ‘l%e biochemical charac&stics of p4OTAK wae similar to the catalytic subunit of proteinkinasec. Herewereportthatinadditiontop4oTAK,pmteia kinaae C (PKC) iaoform was also conatimtively activated in extracts derived from an -ma of the lung. Immunomadivity to PKC
co-eh~tedwithp4OTAK,byMonoQ chromatography. HewsWar, in conuust tcPKC, the activity of p40TAK was not inbiiited when the kinaaeaaaayawaepafamcdinthepfesauxofapaeudwbatrateof PKC. ‘Ikae studies suggest that p4UTAK is diatiwt from PKC.
ActivationofPKC,inthisadcwcarcinoma,raisestheporsibilitytha PKCdepen&nt signal ba&uction tumors of this histological type.
pathways may play a role in lung