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Relationship between the doubling time of hepatocellular carcinoma and the Nucleo-cytoplasmic ratio of cancer cells
April 1995
Relationship Between the Doubling Time of Hepatocellular C a r c i n o m a and the Nucleo-Cytoplasmic Ratio o f Cancer Cells. S. Watanabe, K. Matuda, M. Nishioka. Third Department of Internal Medicine, Kagawa Medical School, Kagawa, Japan. To determine whether the nucleo-cytoplasmic ratio(N/C) of the hepatocellular carcinoma(HCC) cells can be a marker for the grade of malignancy of the HCC, we followed the growth rate of 22 HCC nodules in 22 HCC patients with liver cirrhosis (12 males, 10 females, mean age59.9 years old) foi" one to 31 months ( mean 6.7 months) using ultrasonography( Toshiba SSA 270, Japan). The range of longest diameter of the HCC nodule was 0.8 cm to 3.3 cm (mean-l.8 cm). The N/C was determined by measuring the cell size and nuclear size of the 20 HCC cells in each specimen obtained by needle biopsy or surgical resection. The doubling time of the HCC nodules was calculated by Schwartz's method. The average doubling time (DT) of the HCC nodule was 177 5:199 days. The DT was not correlated with the size of the HCC nodule, cell size nor nuclear size of the HCC. In contrast, the N/C closely correlated with the DT (r=-0.510. p<.05); namely, the larger the value of N/C, the shorter D/F becomes. The HCC specimens were stained by cdc2 kinase (Monoclonal Transduction Laboratories), a cell cycle regulating protein acting at the G1-S and G2-M stages, using the avidinbiotin immunohistochemichal technique. Three out of the 22 cases showed a positive staining for cdc2 in the nucleus of the HCC cells. Positive cells were smaller (p<.01) and had a smaller nucleus (p<.01) compared to the other cdc2 negative cells, however, N/C was not different. The DT of the HCC nodules, including positive cells, was 58 5:24 days. The DT of the HCC nodules, including negative cells, was 201-----226 days. Tt~ difference between the DT of positive and negative cells was significant (p<.05). We made the following conclusions: 1. The doubling time of hepatoceUular carcinoma was correlated with the nucleo-cytoplasmic ratio of the cancer cell; 2. The cdc2 positive cells were smaller and had a smaller nucleus compared to the other cdc2 negative cells. The doubling time of bepatocellular carcinoma nodules, including cdc2 positive cells, was shorter than that of the nodules including cdc2 negative cells.
• CONSERVATION OF INTRACELLULAR pH AFTER COLD ISCHEM1A IN RAT HEPATOCYTES: EFFECT OF DIFFERENT PRESERVATION SOLUTIONS. M Weinlich, C. Baumstark, R. Viababn, M. Starlinger. Surgical Department, University of T0bingen, Germany Preservation solutions, like UW (University of Wisconsin), HTK (Bretschnelder) or EC (Euro Collins), cannot prevent cell damage due to cold ischemia during organ transplantation, even when optimal surgical technique is guaranteed. The goal of this study was to trace non-invasively intracellular pH (PHi) and viability of hepatocytes during simulated cold ischemia. Method: Rat hepatocytes were exposed to different preservation solutions (UW, HTK, EC) for 2, 4 and 6 hours to anoxia at 4°C. Anoxia was achieved by 100% N 2 for UW and HTK, respectively 95% N2/5% CO2 for EC. Cells were loaded with 5pM BCECF-AM, a pH sensitive fluorescent dye, for 30 rain. The cover glass with the loaded cells was mounted in a micro perfusion chamber and viewed throul~, a fluorescent microscope. Repeffusion aRer cold isehemia was accomplished with Krabs-Henselelt, pH 7.4. Viability was traced by BCECF dye distribution. Results: W'Rh increasing duration of cold ischemia, the pHi of the hepatoeytes decreased, independent to tim used preservation solution. In contrast ~idification was d,haendent to the used preservation medium. After 6 hours of cold iselmmia the pHi of the hepatoeytes in UW decreased from 7.40 prior to cold i~hemia to 7.24. In HTK the pHi reached 6.90. Less than 20% of the cells exposed to EC survived after 6 hours of cold ischemia prior to reper~sion, therefore pHi could not reliably be measured in EC. In UW viability was 68°,6 and in HTK' 43% after reperfnsion with 37° C Krabs-Henselelt solution. Cells exposed to UW showed no significant pHi changes after reperfusion. In HTK cells slightly acidified and in EC cells significantly elkalised. Acidification with 20 mmo! .NH4CI after reperfusion let to rapid back regulation of pHi In survwmg cells, indicating adequate intrnceHular regulatory systems, Conclusion: During simulated cold ischemia and warm reperfusion o f rat hepat.oc~,es the le~t., pHi changes could he seen with UW. HTK and EC let to slgniticantly higher pH disturbances and lower mrvival a~er 6 hours of cold ischemia. This model may be used to further optimise preservation solutions.
AASLD
Al197
• HIGH FREQUENCY OF RELAPSE IN PATIENTS WITH AUTOANTIBODIES AND CHRONIC HEPATITIS C IN INTERFERON THERAPY. Y. Watanabe. E. Sanada, H. Moriya, M. Kitamoto, T. Nakanishi, G. Kajiyama. First Depmlment of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan. Patients with chronic hepatitis C frequently have serum autoantibodies. Appearance of autoantibodies is considered to be a kind of clinical indicators of breakdown in immune system. To determine whether existence of autoantibodies influences the efficacy of interferon (IFN) therapy in chronic hepatitis C, we examined the course of serum ALT and hepatitis C virus RNA during treatment and throughout followup for at least 6 mo after therapy discontinuation. 72 patients with chronic hepatitis C treated with IFN were tested for 8 different autoantibodies such as rheumatoid factor, ANA, anti-ssDNA, anti-dsDNA, LE cell, AMA, thyroid autoantibodies, anti-microsomal antibodies prior to IFN therapy in principle. One or more of 8 different autoantibodies were positive in 46 patients, who were classified into autoantibody-positive group. None of 8 different autoantibodies were detected in 26 patients, who were classified into autoantibody-negative group. By the end of IFN therapy the levels of ALTnormalized in 18(69%) of autoantibody-negative group, and 10(39%) maintained normal ALT values throughout the follow-up. On the other hand in autoantibody-positive group, although 24(52%) of them normalized in the levels of ALT by the end of IFN therapy, only 5(11%) maintained normal ALT values during the follow-up due to high rate of relapse after therapy discontinuation. Clearance of viremia was observed in 57% of autoantibody-positive group at the end of IFN therapy, but in 75% of them viremia recurred within 6 months after therapy discontinuation. In autoantibody-negative group sustained response was maintained in 45% of patients with HCV genotype II and 80% of them with genotype III or IV. On the contrary in autoantibody-positive group sustained response was maintained in none of patients with HCV genotype II and 30% of them with genotype III or IV. In patients with high quantity of HCV RNA sustained response was maintained in 30% of autoantibodynegative patients and 8% of autoantibody-positive patients. In conclusion, appearance of autoantibodies predicts low likelihood of sustained response. In host with immunological disorders such as appearance of autoantibodies complete clearance of HCV may be difficult even if clearance of viremia during IFN administration occurs, and such patients are prone to relapse after discontinuation of IFN therapy.
• INCREASED HEPATIC MICROSOMAL CYP 2E 1 PROTEIN IN A RAT NUTRITIONAL MODEL OF NON-ALCOHOLIC STEATOHEPAT1TIS. M.D. Weltman, C. Liddle, M. Murray and G,C. Farrell. Storr Liver Unit, University of Sydney at Westmead Hospital, NSW 2145, Australia. Non-alcoholic steatohepatitis (NASH) produces liver disease that is morphologically identical to alcoholic hepatitis. Hepatic levels of the cytochrome P450 (CYP) 2El are elevated in alcoholic hepatitis. 2El is also increased in obesity and diabetes, disorders associated with NASH in humans. Aim~ To test our hypothesis that 2El could generate reactive oxygen species as tissue-damaging intermediates in NASH, we determined hepatic 2El content and catalytic activity in a rat nutritional model of liver injury. Methods Male Wistar rats were fed a methionine and cholinedeficient diet (MCDD) for 4 or 13 weeks. Controls were fed a nutritionally replete diet. 2E 1 was evaluated in liver sections by immunohistochemistry, and in microsomal fractions by immunoblotting using anti-rat 2El antibody. Catalytic activity of 2E 1 was measured as aniline hydroxylation while other CYP activities (2A1, 3A2, and 2Cll) were determined by pathways of testosterone hydroxylation. Results In MCDD rats, steatohepatitis was confirmed histologically at 4 weeks and was extensive by 13 weeks. Fatty change, an inflammatory infiltrate and hepatocellular necrosis were more prominent in zone 3. Total microsomal P450, and catalytic pathways of testosterone hydroxylation mediated by 2C11 (2cq 16a), 3A2 (61~)and 2A1 (7c0 were reduced in the MCDD groups compared with controls (Table). 16¢~-OHt 2o~-OHt 7or-OH* 6B-OH* P450(%) CONROLS 2.0!-_0.2 1.7+0.1 0.3+0 2.0+0.1 100 MCDD(4W) 0.4+0.1" 0.2+0.1" 0.2+0.0* 1.1+0.1" 70~ MCDD(I3W) 0.4+0.1" 0.3+0.0* 0.2+0.0* 0.9+0.1" 67~ tmean :4-SEM: nmol product/min/mg protein. *p<0.001. ~P<0.05 In contrast, 2El-catalyzed aniline hydroxylase activity was not altered. Hepatic microsomal levels of 2El protein were increased 3-fold at 4 weeks (p<0.000l) and at 13 weeks (p<0.0001). In normal liver, 2El immunostaining was confined to centrilobular hepatocytes. In MCDD liver, 2El staining was increased in intensity and the distribution was altered, corresponding to that of fatty infiltration. Conclusion Rats fed a MCDD diet provide a reliable animal equivalent of NASH. In this model, total P450 and catalytic activities of several CYP proteins were reduced. In contrast, 2El protein was increased and this contributed to the preservation of 2El catalytic activity. The similarities between this experimental model of NASH and alcoholic steatohepatitis lead us to invoke common pathogenetic mech~isms.
Relationship Between the Doubling Time of Hepatocellular C a r c i n o m a and the Nucleo-Cytoplasmic Ratio o f Cancer Cells. S. Watanabe, K. Matuda, M. Nishioka. Third Department of Internal Medicine, Kagawa Medical School, Kagawa, Japan. To determine whether the nucleo-cytoplasmic ratio(N/C) of the hepatocellular carcinoma(HCC) cells can be a marker for the grade of malignancy of the HCC, we followed the growth rate of 22 HCC nodules in 22 HCC patients with liver cirrhosis (12 males, 10 females, mean age59.9 years old) foi" one to 31 months ( mean 6.7 months) using ultrasonography( Toshiba SSA 270, Japan). The range of longest diameter of the HCC nodule was 0.8 cm to 3.3 cm (mean-l.8 cm). The N/C was determined by measuring the cell size and nuclear size of the 20 HCC cells in each specimen obtained by needle biopsy or surgical resection. The doubling time of the HCC nodules was calculated by Schwartz's method. The average doubling time (DT) of the HCC nodule was 177 5:199 days. The DT was not correlated with the size of the HCC nodule, cell size nor nuclear size of the HCC. In contrast, the N/C closely correlated with the DT (r=-0.510. p<.05); namely, the larger the value of N/C, the shorter D/F becomes. The HCC specimens were stained by cdc2 kinase (Monoclonal Transduction Laboratories), a cell cycle regulating protein acting at the G1-S and G2-M stages, using the avidinbiotin immunohistochemichal technique. Three out of the 22 cases showed a positive staining for cdc2 in the nucleus of the HCC cells. Positive cells were smaller (p<.01) and had a smaller nucleus (p<.01) compared to the other cdc2 negative cells, however, N/C was not different. The DT of the HCC nodules, including positive cells, was 58 5:24 days. The DT of the HCC nodules, including negative cells, was 201-----226 days. Tt~ difference between the DT of positive and negative cells was significant (p<.05). We made the following conclusions: 1. The doubling time of hepatoceUular carcinoma was correlated with the nucleo-cytoplasmic ratio of the cancer cell; 2. The cdc2 positive cells were smaller and had a smaller nucleus compared to the other cdc2 negative cells. The doubling time of bepatocellular carcinoma nodules, including cdc2 positive cells, was shorter than that of the nodules including cdc2 negative cells.
• CONSERVATION OF INTRACELLULAR pH AFTER COLD ISCHEM1A IN RAT HEPATOCYTES: EFFECT OF DIFFERENT PRESERVATION SOLUTIONS. M Weinlich, C. Baumstark, R. Viababn, M. Starlinger. Surgical Department, University of T0bingen, Germany Preservation solutions, like UW (University of Wisconsin), HTK (Bretschnelder) or EC (Euro Collins), cannot prevent cell damage due to cold ischemia during organ transplantation, even when optimal surgical technique is guaranteed. The goal of this study was to trace non-invasively intracellular pH (PHi) and viability of hepatocytes during simulated cold ischemia. Method: Rat hepatocytes were exposed to different preservation solutions (UW, HTK, EC) for 2, 4 and 6 hours to anoxia at 4°C. Anoxia was achieved by 100% N 2 for UW and HTK, respectively 95% N2/5% CO2 for EC. Cells were loaded with 5pM BCECF-AM, a pH sensitive fluorescent dye, for 30 rain. The cover glass with the loaded cells was mounted in a micro perfusion chamber and viewed throul~, a fluorescent microscope. Repeffusion aRer cold isehemia was accomplished with Krabs-Henselelt, pH 7.4. Viability was traced by BCECF dye distribution. Results: W'Rh increasing duration of cold ischemia, the pHi of the hepatoeytes decreased, independent to tim used preservation solution. In contrast ~idification was d,haendent to the used preservation medium. After 6 hours of cold iselmmia the pHi of the hepatoeytes in UW decreased from 7.40 prior to cold i~hemia to 7.24. In HTK the pHi reached 6.90. Less than 20% of the cells exposed to EC survived after 6 hours of cold ischemia prior to reper~sion, therefore pHi could not reliably be measured in EC. In UW viability was 68°,6 and in HTK' 43% after reperfnsion with 37° C Krabs-Henselelt solution. Cells exposed to UW showed no significant pHi changes after reperfusion. In HTK cells slightly acidified and in EC cells significantly elkalised. Acidification with 20 mmo! .NH4CI after reperfusion let to rapid back regulation of pHi In survwmg cells, indicating adequate intrnceHular regulatory systems, Conclusion: During simulated cold ischemia and warm reperfusion o f rat hepat.oc~,es the le~t., pHi changes could he seen with UW. HTK and EC let to slgniticantly higher pH disturbances and lower mrvival a~er 6 hours of cold ischemia. This model may be used to further optimise preservation solutions.
AASLD
Al197
• HIGH FREQUENCY OF RELAPSE IN PATIENTS WITH AUTOANTIBODIES AND CHRONIC HEPATITIS C IN INTERFERON THERAPY. Y. Watanabe. E. Sanada, H. Moriya, M. Kitamoto, T. Nakanishi, G. Kajiyama. First Depmlment of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan. Patients with chronic hepatitis C frequently have serum autoantibodies. Appearance of autoantibodies is considered to be a kind of clinical indicators of breakdown in immune system. To determine whether existence of autoantibodies influences the efficacy of interferon (IFN) therapy in chronic hepatitis C, we examined the course of serum ALT and hepatitis C virus RNA during treatment and throughout followup for at least 6 mo after therapy discontinuation. 72 patients with chronic hepatitis C treated with IFN were tested for 8 different autoantibodies such as rheumatoid factor, ANA, anti-ssDNA, anti-dsDNA, LE cell, AMA, thyroid autoantibodies, anti-microsomal antibodies prior to IFN therapy in principle. One or more of 8 different autoantibodies were positive in 46 patients, who were classified into autoantibody-positive group. None of 8 different autoantibodies were detected in 26 patients, who were classified into autoantibody-negative group. By the end of IFN therapy the levels of ALTnormalized in 18(69%) of autoantibody-negative group, and 10(39%) maintained normal ALT values throughout the follow-up. On the other hand in autoantibody-positive group, although 24(52%) of them normalized in the levels of ALT by the end of IFN therapy, only 5(11%) maintained normal ALT values during the follow-up due to high rate of relapse after therapy discontinuation. Clearance of viremia was observed in 57% of autoantibody-positive group at the end of IFN therapy, but in 75% of them viremia recurred within 6 months after therapy discontinuation. In autoantibody-negative group sustained response was maintained in 45% of patients with HCV genotype II and 80% of them with genotype III or IV. On the contrary in autoantibody-positive group sustained response was maintained in none of patients with HCV genotype II and 30% of them with genotype III or IV. In patients with high quantity of HCV RNA sustained response was maintained in 30% of autoantibodynegative patients and 8% of autoantibody-positive patients. In conclusion, appearance of autoantibodies predicts low likelihood of sustained response. In host with immunological disorders such as appearance of autoantibodies complete clearance of HCV may be difficult even if clearance of viremia during IFN administration occurs, and such patients are prone to relapse after discontinuation of IFN therapy.
• INCREASED HEPATIC MICROSOMAL CYP 2E 1 PROTEIN IN A RAT NUTRITIONAL MODEL OF NON-ALCOHOLIC STEATOHEPAT1TIS. M.D. Weltman, C. Liddle, M. Murray and G,C. Farrell. Storr Liver Unit, University of Sydney at Westmead Hospital, NSW 2145, Australia. Non-alcoholic steatohepatitis (NASH) produces liver disease that is morphologically identical to alcoholic hepatitis. Hepatic levels of the cytochrome P450 (CYP) 2El are elevated in alcoholic hepatitis. 2El is also increased in obesity and diabetes, disorders associated with NASH in humans. Aim~ To test our hypothesis that 2El could generate reactive oxygen species as tissue-damaging intermediates in NASH, we determined hepatic 2El content and catalytic activity in a rat nutritional model of liver injury. Methods Male Wistar rats were fed a methionine and cholinedeficient diet (MCDD) for 4 or 13 weeks. Controls were fed a nutritionally replete diet. 2E 1 was evaluated in liver sections by immunohistochemistry, and in microsomal fractions by immunoblotting using anti-rat 2El antibody. Catalytic activity of 2E 1 was measured as aniline hydroxylation while other CYP activities (2A1, 3A2, and 2Cll) were determined by pathways of testosterone hydroxylation. Results In MCDD rats, steatohepatitis was confirmed histologically at 4 weeks and was extensive by 13 weeks. Fatty change, an inflammatory infiltrate and hepatocellular necrosis were more prominent in zone 3. Total microsomal P450, and catalytic pathways of testosterone hydroxylation mediated by 2C11 (2cq 16a), 3A2 (61~)and 2A1 (7c0 were reduced in the MCDD groups compared with controls (Table). 16¢~-OHt 2o~-OHt 7or-OH* 6B-OH* P450(%) CONROLS 2.0!-_0.2 1.7+0.1 0.3+0 2.0+0.1 100 MCDD(4W) 0.4+0.1" 0.2+0.1" 0.2+0.0* 1.1+0.1" 70~ MCDD(I3W) 0.4+0.1" 0.3+0.0* 0.2+0.0* 0.9+0.1" 67~ tmean :4-SEM: nmol product/min/mg protein. *p<0.001. ~P<0.05 In contrast, 2El-catalyzed aniline hydroxylase activity was not altered. Hepatic microsomal levels of 2El protein were increased 3-fold at 4 weeks (p<0.000l) and at 13 weeks (p<0.0001). In normal liver, 2El immunostaining was confined to centrilobular hepatocytes. In MCDD liver, 2El staining was increased in intensity and the distribution was altered, corresponding to that of fatty infiltration. Conclusion Rats fed a MCDD diet provide a reliable animal equivalent of NASH. In this model, total P450 and catalytic activities of several CYP proteins were reduced. In contrast, 2El protein was increased and this contributed to the preservation of 2El catalytic activity. The similarities between this experimental model of NASH and alcoholic steatohepatitis lead us to invoke common pathogenetic mech~isms.