- Email: [email protected]
Role of adenosine receptors in the inhibition of ifbroproliferation due to pentoxifylline and M-1 metabolite
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995
AASLD ABSTRACTS
1473 ROLE OF ADENOSINE RECEPTORS IN THE INHIBITION OF
1474 ANALYSIS OF THE T CELL ANTIGEN RECEPTOR GENE
REPERTOIRE IN AUTOIMMUNE LIVER DISEASE John O. Phillins. Weizhon~ Yin~ and James F. George. Division of Gastroenterology/Hepatolog-y& I:)epartment of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-0007 Autoimmune hepatitis (AH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic liver diseases in which the presence of T lymphocyte infiltrates and liver cell destruction support the hypothesis that an autoimmune response is a significant component in the pathogenesis of these diseases. Limited expression of T cell antigen receptor (TCR) variable region gene segment families has been observed in several organ-specific autoimmune diseases including rheumatoid arthritis, thyroiditis and multiple sclerosis supporting the hypothesis that an antigen driven expansion of infiltrating T cells plays a pathgenic role in these disorders. Therefore, the AIM of this study was to investigate the utilization of TCR I~-chain variable region gene segment families in patients with chronic autoimmune liver disease. METHODS: RNA was isolated from liver tissue obtained from patients undergoing orthotopic liver transplantation for AH (N = 3), PBC (N = 2) or PSC (N = 1). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify transcripts bearing the 22 V~ gene segment families isolated from explanted liver. RNA isolated from peripheral blood mononuclear ceils were used a control. RESULTS: No restriction of TCR VI~expression was observed by RT-PCR in liver from patients with autoimmune liver disease. At least 19 of the 22 Vi~ gene segment families were detected in patients with autoimmune liver disease. By comparison, 21 of the 22 VI~ gene segment families were detected by RT-PCR in RNA from peripheral blood mononuclear cells CONCLUSION: TCR VI~ gene segment expression in affected livers was not restricted in autoimmune liver diseases including PBC, PSC or AH. Unresolved issues in this study which may influence these results include the role of contaminating peripheral blood that remains in the explanted liver and whether changes in lymphocyte infiltrates occur over the course of these chronic liver diseases.
FIBROPROLIFERATION DUE TO PENTOXIFYLLINE AND M-1 METABOLITE. T.C. Peterson and B. White. Depts. of Medicine and Pharmacology, Dalhousie University, Halifax, N.S., Canada. Previous results indicate that pentoxifylline blocks both the biochemical and histological changes characteristic of fibrosis in a swine model of hepatic fibrosis. Pentoxifylline, it's M1 metabolite 3,7dimethyl-l-(5-hydroxyhexyl)xanthine and adenosine inhibit platelet derived growth factor (PDGF) stimulated fibroproliferation in a dose related manner. Fibroproliferation is assessed by 3H-thymidine uptake, cell count and MTT assay. To determine the role of adenosine receptors in the inhibition of fibroproliferation that is observed with pentoxifylline a non selective adenosine receptor antagonist 8-phenyltheophylline (8-PT) was used. A 2 adenosine receptors are found on fibroblasts and may mediate inhibition of fibroproliferation which occurs with pentoxifylline. Pentoxifylline has been reported to inhibit adenosine reuptake thus potentiating actions of adenosine. Pretreatment of fibroblasts with 8-phenyltheophylline prior to addition of pentoxifyUine may prevent the actions of pentoxifylline. To test this hypothesis, the effect of 8-phenyltheophylline was assessed at 1, 10, 50 and 100/~M. The results indicate that pretreatment of fibroblasts with 8-phenyltheophylline did not alter the inhibitory effect of pentoxifylline on PDGF (8ng/ml) stimulated fibroproliferation. 8-PT also did not alter the inhibitory effect of pentoxifylline on baseline proliferation of fibroblasts. 8-PT did not prevent the inhibitory effect of the M1 metabolite. The lack of effect of 8-PT on the pentoxifylline mediated inhibition would argue against a mechanism involving adenosine receptors thus inhibition of adenosine reuptake as the mechanism for pentoxifylline's effect in this system is unlikely. (supported by the Medical Research Council of Canada).
1475 TAMOXIFEN INHIBITS THE GROWTH OF TWO HUMAN CHOLANGIOCARCINOMA CELL LINES John O. Phillins. Weizhon~ Yin~, and Selwvn Vickers. Division of Gastroenterology/Hepatology & Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-0007 Cholangiocarcinoma is a biliary epithelial malignancy which is almost universally fatal when it occurs at the hilar bifurcation. The poor prognosis reflects a lack of effective medical or surgical therapy, the absence of early detection methods and inadequate chemotherapeutic or radiation adjuvant therapy. Tamoxifen is an effective, well tolerated agent useful as adjunctive therapy for breast cancer. Recently, tamoxifen has been shown to be effective in the treatment of other malignancies including tumors of the central nervous system and hepatocellnlar carcinoma. The AIM of the current study was to investigate the in vitro response of two different human cholangiocareinoma cell lines to the antiestrogenic agent, tamoxifen. METHODS: Two established human cholangiocarcinoma cell lines (Mz-Cha-1 and OZ) which maintain their biliary phenotype as determined by CK-19 and .t-glutamyltranspeptidase activity were maintained as adherent epithelial cells in culture. Cell lines were stained for estrogen and progesterone receptors by indirect immunocytochemistry. For inhibition studies, cell lines were seeded into plates (5 x 104 to 1.5 x 105 cells/well) and effects of tamoxifen on cell growth rates were determined over a wide dose range of tamoxifen (0.1-25 I.tM). Cell counting was performed with an automated cell counter and viability determined by trypan blue exclusion. RESULTS: Doubling times of the two tumor lines differed: ~30 hours for Mz-Cha-1 and -48 hours for OZ. Tamoxifen caused a dose dependent inhibition of both Mz-Cha-1 and OZ tumor lines. At a concentration of 2.5-5.0 I.tM, tamoxifen was effective in causing >50% inhibition of tumor cell growth. This dose is within the range reported in the serum of patients receiving long term treatment with tamoxifen. Cell viability was unaffected. Estrogen and progesterone receptors were not detected by immunocytochemistry on either cell line. CONCLUSION: The in vitro growth of two separate human cholangiocarcinoma cell lines was markedly inhibited by tamoxifen. Tamoxifen may represent a potential therapeutic alternative for the treatment of cholangiocarcinoma.
475A
1476
HISTOLOGICAL EVALUATION OF LIVER FIBROSIS : OBSERVER SCORE OR IMAGE ANALYSIS ? Pilette ~, Rousselet MC. Bedossa P. ChaDDard D. Oberti F. Ma~aa ~, Gallols Y. CalAs p. CHU Angers and Kremlin-Bic~tre, France. Liver fibrosis is one of the m a i n c o n s e q u e n c e s of chronic liver d i s e a s e s (CLD). Until now, it was e v a l u a t e d by q u a l i t a t i v e h i s t o l o g i c a l e x a m i n a t i o n . Recently, were introduced semi-quantitative histological scores or q u a n t i t a t i v e determination using image analysis. However, these latter methods have not b e e n v a l i d a t e d . Therefore, we have prospectively s t u d i e d t h e s e 2 m e t h o d s in 83 patients with CLD. The histological score of f i b r o s i s was e v a l u a t e d by 2 p a t h o l o g i s t s u s i n g a score with 7 grades ; the fibrosis area was measured by a Leica device after picrosirius staining of fibrosis in liver biopsies. The f o l l o w i n g s e r u m m a r k e r s of f i b r o s i s were m e a s u r e d : h y a l u r o n a t e , PIIIP, ~2 m a c r o g l o b u l i n , PGA score and p r o t h r o m b i n index (PI). The f i b r o s i s area was 11±8 % in non c i r r h o t i c p a t i e n t s a n d 33±10 % in cirrhotic patients (P<10-4). T h e r e was a g o o d c o r r e l a t i o n b e t w e e n the h i s t o l o g i c a l score and the fibrosis area (r=0.84, p<10-4). The highest correlations of h i s t o l o g i c a l score were obtained with h y a l u r o n a t e (r=0.78, p<10 -4) and PI (r=-0.72, P
AASLD ABSTRACTS
1473 ROLE OF ADENOSINE RECEPTORS IN THE INHIBITION OF
1474 ANALYSIS OF THE T CELL ANTIGEN RECEPTOR GENE
REPERTOIRE IN AUTOIMMUNE LIVER DISEASE John O. Phillins. Weizhon~ Yin~ and James F. George. Division of Gastroenterology/Hepatolog-y& I:)epartment of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-0007 Autoimmune hepatitis (AH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic liver diseases in which the presence of T lymphocyte infiltrates and liver cell destruction support the hypothesis that an autoimmune response is a significant component in the pathogenesis of these diseases. Limited expression of T cell antigen receptor (TCR) variable region gene segment families has been observed in several organ-specific autoimmune diseases including rheumatoid arthritis, thyroiditis and multiple sclerosis supporting the hypothesis that an antigen driven expansion of infiltrating T cells plays a pathgenic role in these disorders. Therefore, the AIM of this study was to investigate the utilization of TCR I~-chain variable region gene segment families in patients with chronic autoimmune liver disease. METHODS: RNA was isolated from liver tissue obtained from patients undergoing orthotopic liver transplantation for AH (N = 3), PBC (N = 2) or PSC (N = 1). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify transcripts bearing the 22 V~ gene segment families isolated from explanted liver. RNA isolated from peripheral blood mononuclear ceils were used a control. RESULTS: No restriction of TCR VI~expression was observed by RT-PCR in liver from patients with autoimmune liver disease. At least 19 of the 22 Vi~ gene segment families were detected in patients with autoimmune liver disease. By comparison, 21 of the 22 VI~ gene segment families were detected by RT-PCR in RNA from peripheral blood mononuclear cells CONCLUSION: TCR VI~ gene segment expression in affected livers was not restricted in autoimmune liver diseases including PBC, PSC or AH. Unresolved issues in this study which may influence these results include the role of contaminating peripheral blood that remains in the explanted liver and whether changes in lymphocyte infiltrates occur over the course of these chronic liver diseases.
FIBROPROLIFERATION DUE TO PENTOXIFYLLINE AND M-1 METABOLITE. T.C. Peterson and B. White. Depts. of Medicine and Pharmacology, Dalhousie University, Halifax, N.S., Canada. Previous results indicate that pentoxifylline blocks both the biochemical and histological changes characteristic of fibrosis in a swine model of hepatic fibrosis. Pentoxifylline, it's M1 metabolite 3,7dimethyl-l-(5-hydroxyhexyl)xanthine and adenosine inhibit platelet derived growth factor (PDGF) stimulated fibroproliferation in a dose related manner. Fibroproliferation is assessed by 3H-thymidine uptake, cell count and MTT assay. To determine the role of adenosine receptors in the inhibition of fibroproliferation that is observed with pentoxifylline a non selective adenosine receptor antagonist 8-phenyltheophylline (8-PT) was used. A 2 adenosine receptors are found on fibroblasts and may mediate inhibition of fibroproliferation which occurs with pentoxifylline. Pentoxifylline has been reported to inhibit adenosine reuptake thus potentiating actions of adenosine. Pretreatment of fibroblasts with 8-phenyltheophylline prior to addition of pentoxifyUine may prevent the actions of pentoxifylline. To test this hypothesis, the effect of 8-phenyltheophylline was assessed at 1, 10, 50 and 100/~M. The results indicate that pretreatment of fibroblasts with 8-phenyltheophylline did not alter the inhibitory effect of pentoxifylline on PDGF (8ng/ml) stimulated fibroproliferation. 8-PT also did not alter the inhibitory effect of pentoxifylline on baseline proliferation of fibroblasts. 8-PT did not prevent the inhibitory effect of the M1 metabolite. The lack of effect of 8-PT on the pentoxifylline mediated inhibition would argue against a mechanism involving adenosine receptors thus inhibition of adenosine reuptake as the mechanism for pentoxifylline's effect in this system is unlikely. (supported by the Medical Research Council of Canada).
1475 TAMOXIFEN INHIBITS THE GROWTH OF TWO HUMAN CHOLANGIOCARCINOMA CELL LINES John O. Phillins. Weizhon~ Yin~, and Selwvn Vickers. Division of Gastroenterology/Hepatology & Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-0007 Cholangiocarcinoma is a biliary epithelial malignancy which is almost universally fatal when it occurs at the hilar bifurcation. The poor prognosis reflects a lack of effective medical or surgical therapy, the absence of early detection methods and inadequate chemotherapeutic or radiation adjuvant therapy. Tamoxifen is an effective, well tolerated agent useful as adjunctive therapy for breast cancer. Recently, tamoxifen has been shown to be effective in the treatment of other malignancies including tumors of the central nervous system and hepatocellnlar carcinoma. The AIM of the current study was to investigate the in vitro response of two different human cholangiocareinoma cell lines to the antiestrogenic agent, tamoxifen. METHODS: Two established human cholangiocarcinoma cell lines (Mz-Cha-1 and OZ) which maintain their biliary phenotype as determined by CK-19 and .t-glutamyltranspeptidase activity were maintained as adherent epithelial cells in culture. Cell lines were stained for estrogen and progesterone receptors by indirect immunocytochemistry. For inhibition studies, cell lines were seeded into plates (5 x 104 to 1.5 x 105 cells/well) and effects of tamoxifen on cell growth rates were determined over a wide dose range of tamoxifen (0.1-25 I.tM). Cell counting was performed with an automated cell counter and viability determined by trypan blue exclusion. RESULTS: Doubling times of the two tumor lines differed: ~30 hours for Mz-Cha-1 and -48 hours for OZ. Tamoxifen caused a dose dependent inhibition of both Mz-Cha-1 and OZ tumor lines. At a concentration of 2.5-5.0 I.tM, tamoxifen was effective in causing >50% inhibition of tumor cell growth. This dose is within the range reported in the serum of patients receiving long term treatment with tamoxifen. Cell viability was unaffected. Estrogen and progesterone receptors were not detected by immunocytochemistry on either cell line. CONCLUSION: The in vitro growth of two separate human cholangiocarcinoma cell lines was markedly inhibited by tamoxifen. Tamoxifen may represent a potential therapeutic alternative for the treatment of cholangiocarcinoma.
475A
1476
HISTOLOGICAL EVALUATION OF LIVER FIBROSIS : OBSERVER SCORE OR IMAGE ANALYSIS ? Pilette ~, Rousselet MC. Bedossa P. ChaDDard D. Oberti F. Ma~aa ~, Gallols Y. CalAs p. CHU Angers and Kremlin-Bic~tre, France. Liver fibrosis is one of the m a i n c o n s e q u e n c e s of chronic liver d i s e a s e s (CLD). Until now, it was e v a l u a t e d by q u a l i t a t i v e h i s t o l o g i c a l e x a m i n a t i o n . Recently, were introduced semi-quantitative histological scores or q u a n t i t a t i v e determination using image analysis. However, these latter methods have not b e e n v a l i d a t e d . Therefore, we have prospectively s t u d i e d t h e s e 2 m e t h o d s in 83 patients with CLD. The histological score of f i b r o s i s was e v a l u a t e d by 2 p a t h o l o g i s t s u s i n g a score with 7 grades ; the fibrosis area was measured by a Leica device after picrosirius staining of fibrosis in liver biopsies. The f o l l o w i n g s e r u m m a r k e r s of f i b r o s i s were m e a s u r e d : h y a l u r o n a t e , PIIIP, ~2 m a c r o g l o b u l i n , PGA score and p r o t h r o m b i n index (PI). The f i b r o s i s area was 11±8 % in non c i r r h o t i c p a t i e n t s a n d 33±10 % in cirrhotic patients (P<10-4). T h e r e was a g o o d c o r r e l a t i o n b e t w e e n the h i s t o l o g i c a l score and the fibrosis area (r=0.84, p<10-4). The highest correlations of h i s t o l o g i c a l score were obtained with h y a l u r o n a t e (r=0.78, p<10 -4) and PI (r=-0.72, P