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Activation of NF-κB in non-parenchymal liver cells after partial hepatectomy in rat: possible involvement in HB-EGF expression
HEPATOLOGYVo1. 34, No. 4, Pt. 2, 2 0 0 1
AASLD ABSTRACTS
391A
875
876
THE ABSENCE OF UP-REGULATION OF TELOMERASE ACTIVITY DURING REGENERATION AFTER PARTIAL HEPATECTOMY IN HBV X GENE TRANSGENIC MICE. Hiroshige Kojima, Kelly D Kaita, Univ of Manitoba, Winnipeg, MB Canada; Z h e n m i n g Xu, James H On, Univ of Southern California, Los Angeles, CA; Yuewen Gong, Gerald Y Minuk, Univ of Manitoba, Winnipeg, MB Canada
ACTIVATION OF NF-KB IN NON-PARENCHYMAL LIVER CELLS AFTER PARTIAL HEPATECTOMY IN RAT: POSSIBLE INVOLVEMENT IN HBEGF EXPRESSION. Shigern Sakuda, Sinji Tamura, Akira Yamada, Jun-lchiro Miyagawa, Koji Yamamoto, Shinichi Kiso, N o b u y u k i ho, Shigeki Higashiyama, Naoyuki Taniguti, Sumio Kawata, Yuji Matsuzawa, Grad Sch of Medicine, Osaka Univ, Suita J a p a n
Background: Hepatocellular carcinoma (HCC) is a c o m m o n t u m o r in patients with chronic hepatitis B virus (HBV) infections. Transgenic mice that express the HBV X protein (HBx) have previously been s h o w n to be more sensitive to the effects of hepatocarcinogens, a l t h o u g h the m e c h a n i s m for this finding remains to be determined. Telomerase activity, the enzyme responsible for maintaining telomere length a n d t h e r e b y c h r o m o s o m a l stability, is absent in most tissues. However, very h i g h levels h a v e been detected in various malignancies including HCC. In rodents, hepatic telomerase activity increases in regenerating livers (Tsujiuchi et al., Cancer Lett. 122: 115-120, 1998). This increase m a y serve to protect hepatocyte DNA from c h r o m o s o m a l instability. In the present study, we hypothesized that the expression of HBx m i g h t interfere with increases in telomerase activity a n d thereby, render these cells more susceptible to carcinogenic mutations. Methods: Eight-week-old CD-1 male mice were obtained from Charles River Laboratories. Two-thirds partial hepatectomies (PHx) were performed by the m e t h o d of Higgins a n d Anderson u n d e r ether anesthesia after fasting for 12 hours. Groups of four to six mice were killed at 0, 6, 12, 24, 36, 48 h o u r s a n d their livers removed a n d snap frozen in liquid nitrogen. Telomerase activity was semi-quantitatively measured by Telomerase PCR ELISA PLus (Roche Diagnostics). F o u r HBx transgenic mice were killed at 12 h o u r s post-PHx w h e n m a x i m u m telomerase activity was observed in the CD-1 non-transgenic mice. mRNA of the telomerase catalytic subunit; mice telomerase reverse transcriptase (mTERT), was q u a n titatively measured by real-time RT-PCR with LightCycler (Roche Diagnostics). Results: Telomerase activity peaked at 12 h o u r s post-PHx in normal mice (baseline; 1.00 ± 0.23 versus 12 h post-PHx; 1.68 - 0.49, p < 0 . 0 5 ) . However, in HBx transgenic mice, telomerase activity was significantly lower, both at baseline (0.71 ± 0.08, p < 0 . 0 5 ) a n d 12 h post-PHx (0.81 -+ 0.09, p < 0 . 0 1 ) . Following PHx, mTERT mRNA expression remained constant in normal mice. In HBx transgenic mice, c o m p a r e d to baseline, mTERT mRNA expression was significantly decreased at 12 h post-PHx (baseline; 2.69 -+ 0.85 versus 12 h; 0.65 -+ 0.29 X 106 copies//zg, p < 0 . 0 1 ) . Conclusion: Hepatic telomerase activity increases following PH in n o r m a l mice. However, in HBx transgenic mice, the increase in telomerase activity is markedly attenuated as is mTERT mRNA expression. These results suggest that HBx expression m a y play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.
Aims: Several transcription factors have been implicated in the initiation of liver g r o w t h after PH. In particular, nuclear factor (NF) KB is activated very rapidly after PH a n d this activation coincides with the transcriptional induction of immediate-earfy g r o w t h response genes. However, the issue of specificafly where NF-KB is activated is unclear. W e previously reported that heparinbinding epidermal growth factor-like g r o w t h factor (HB-EGF) is a hepatotrophic factor. The aims of this s t u d y were to identify NF-KB-activated cells a n d clarify their involvement in HB-EGF expression after PH. Methods: Using rats, a two-thirds PH was performed, after w h i c h NF-KB-activated ceils a n d HB-EGF-positive cells were identified b y Southwestern histochemistry or immunohistochemistry. NF-KB binding activity a n d HB-EGF gene expression in rat hepatocytes a n d n o n - p a r e n c h y m a l cells (NPC) in primary culture were analyzed by electrophoretic mobility shift assay (EMSA) a n d reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results: Southwestern histochemistry for NF-KB a n d i m m u n o h i s t o c h e m i s t r y showed that the first activated-NF-KB-positive cells were Kupffer cells a n d sinusoidal endothelial cells, w h i c h were detectable at 0.5 h after PH. The hepatocyte nuclei became positive for NF-KB a n d the n u m b e r of activated-NF-KB-positive cells reached a m a x i m u m at 1 h after PH. HB-EGF immunoreactivity became to be detected predominantly in these sinusoidal ceils of the periportal zones at 4.5 h after PH, a n d the localization then extended to all lobules at 12 h after PH. HB-EGF gene expression was accompanied by an increase in NF-KB binding activity in NPC in primary culture, w h i c h was abolished b y pyrrolidine dithiocarbamate (PDTC), a n inhibitor of NF-KB activation. Conclusions: NF-KB was activated in NPC prior to activation in hepatocytes. NF-KB activation m a y be involved in HB-EGF expression in N P C after PH.
877
878
DELAYED LIVER REGENERATION W I T H NEGATIVE REGULATION OF HGF AND POSITIVE REGULATION OF TGF-181 MRNA AFTER PORTAL BRANCH LIGATION IN BILIARY OBSTRUCTED RAT. Toshihisa Yamano, Ryuji Hirai, Shinji Hato, Tadahiro Uemura, Nobuyoshi Shimizu, O k a y a m a Univ Sch of Medicine, O k a y a m a J a p a n
PKR LEVELS ARE INCREASED IN HEPATITIS C VIRUS-RELATED HEPATOCELLULAR CARCINOMA. Yoichi Hiasa, Yoshitaka Kamegaya, Ana M Contreras, W e n p i n g He, Massachusetts General Hospital a n d Harvard Medical School, Boston, MA; Emmett V Schmidt, Massachusetts General Hospital Cancer Center, Boston, MA; R a y m o n d T Chung, Massachusetts General Hospital a n d Harvard Medical School, Boston, MA
Introduction: Extended hepateetomy occasionallycarries with it severe post operative liver failure with high morbidity and mortality rates. Preoperative portal branch embolization, which is a modification of portal branch ligation (PBL), induces hypertrophy of remaining non-emhofized hepatic lobe and atrophy ofembolized hepatic lobe to be reseeted. Therefore, this procedure makes extended hepatectomy safer and minimizes postoperative liver dysfunction. However,obstructive jaundice, which may affect the hepatocyte proliferation, has been observed preoperativelyin some cases, such as with hilar cholangiocarcinoma.Biliaryobstruction is known to induce liver fibrosis in association with the up-regulation of intrahepatic transforming growth factor bets (TGF-]3). TGF-~3inhibits the proliferation of hepatocytes, and exerts stimulatory effects on fibrogenesisby activating hepatic stenate cells (HSCs). Activated HSCs produce a large amount o[ TGF-~ and express the alpha-smooth muscle actin (a-SMA) isoform. On the other hand, hepatocyte growth factor (HGF) is produced chiefly from HSCsand acts as the most potent mitogen on hepatoeytes. However, HGF mRNA expression is lost once HSCs transform into myofibroblast-likecells. Although several studies regarding the liver regeneration after partial hepatectomy or PBL with obstructivejaundice have been performed, it is not clear how the obstructive jaundice affects the liver regeneration after those treatment. Moreover, there were few studies regarding the expression of growth factors in the regenerating liver after those treatments with obstructivejaundice. In this study, we evaluated the effect of obstructive jaundice on liver regeneration after PBL, and the expression of HGF and TGF-181mRNAafter PBLin jaundiced rat liver. Methods: MaleWistsr rats were used. In BDPVLgroup, common bile duct was Iigated,and 7 days later (Day0) the left branch of portal vein, feeding left median and lateral lobes, was ligated. In PVL group, common bile duct was exposed, but not ligated, and 7 days later (Day 0), rats underwent only PBL.After PBL,rats were sacrificed at various times. Liver wet weight, proliferating cell nuclear antigen (PCNA) labeling, HGF and TGF-]31 mRNA expression, and immunohistochemicaI staining with ~-SMA antibody were studied. Results: In the BDPVL and PVL groups, the wet weight ratio of the non-ligated lobe to the whole liver increased prompdy after Day 0. In the PVLgroup, the ratio on Day 5 increased to 81-+1% and was significantly higher than the BDPVLgroup ratio (58_+3%). However,by Day 10, the ratio in BDPVLgroup increased to 79_+3%,close to the PVLgroup ratio (83±3%) at that time. The PCNA labeling index in the non-ligated lobe significantly increased after Day 0 and reached a peak at Day I in both the BDPVLand the PVL groups. The peak level, however, was significantlylower in the BDPVLgroup (19 ~ 1%) than in the pVL group (31±4%). The expression of HGF mRNAremarkably increased after Day 0 in the non-figated growing lobe and the peak was observed at 12 hours in both groups. However,the peak levelin the BDPVLgroup was significantlylower than in the PVL group. The expression of TGF-,81also increased between Day 2 and 5 in the non-ligated lobe of the BDPVLand PVL groups, and the level in the BDPVL group was significantlyhigher than in the PVLgroup, a-SMAstaining showed that hepatic stellate cells (HSCs) were gradually activated into myofibroblast-likecells in the BDPVLgroup. Conclusion: This study has shown that obstructivejaundice has a strong effect on the expression of HGF and TGFq81 mRNA,which are regulated in the negative direction for liver regeneration, resulting in delayed liver regeneration. Furthermore, this study has shown that bfliary obstruction activates HSCs. These findings suggest that HSCs may play an important role in the regulation of the expression of HGF and TGF-/31mRNAin the jaundiced rat liver.
Background/Aims: Hepatitis C virus (HCV) is a leading cause of hepatocellular carcinoma (HCC) worldwide. The double-stranded RNA-activated protein kinase (PKR) mediates the phosphorylation of eukaryotic translation initiation factor elF2-a, w h i c h m a y in turn inhibit HCV protein synthesis. In addition to its putative effects o n viral protein synthesis, PKR also inhibits host cell growth a n d proliferation a n d has therefore been p r o p o s e d to act as a t u m o r suppressor. Recent reports that the HCV NS5A protein tightly binds a n d inhibits PKR activity 1 a n d that HCV E2 inhibits PKR kinase function 2 raise the possibility that HCV contributes to HCC t h r o u g h alterations in PKR. In order to clarify the m e c h a n i s m of HCV-related hepatocarcinogenesis, we examined the expression of PKR in paired malignant a n d non-malignant tissue from patients with HCV-related HCC. In addition, we also sought to determine whether mutations in PKR sequences could be identified from these tissues. Methods: We prepared two sets of primers flanking the PKR E2- a n d the PKR NS5Abinding sites, respectively. Tissue samples were obtained from 9 HCV-infected patients with cirrhosis a n d H C C w h o had u n d e r g o n e liver transplantation or resection. T u m o r (T) a n d s u r r o u n d i n g n o n t u m o r o u s (NT) cirrhotic tissue was extracted for RNA a n d protein. W e performed semiqnantitative RT-PCR using GAPDH as a housekeeping control to determine the relative expression of PKR mRNA between T a n d NT tissue. In addition, TOPO cloning was performed on each cDNA amplicon a n d individual transformants were sequenced (5-10 clones per amplicon). Western blot was performed using pofyclonal antisera for PKR. Results: W e found no significant differences in levels of PKR mRNA between T and NT pairs for either the E2- or NS5A-binding regions. W h e n sequences of individual clones corresponding to each mRNA amplicon were analyzed, we found no mutations in either T or NT tissue. However, PKR protein levels were significantly increased in T compared to NT in all 9 pairs (range 1.2-4.3 fold, mean 2.6, p = 0 . 0 0 0 1 ) . Conclusion: While there are no significant differences in PKR gene expression or sequences between T a n d NT tissue in HCV-related HCC, levels of PKR protein are significantly elevated in HCC tissue. These data suggest that PKR expression is altered at the posttranscriptional level in h u m a n HCC. The finding of increased levels of PKR protein in malignant tissue raises the possibility that PKR does not function as a classical t u m o r suppressor protein driving the formation of HCC. Reference: ZGale M Jr., et al Virology 230 217, 1997.2Taylor DR, et al. Science 285: 107, 1999.
AASLD ABSTRACTS
391A
875
876
THE ABSENCE OF UP-REGULATION OF TELOMERASE ACTIVITY DURING REGENERATION AFTER PARTIAL HEPATECTOMY IN HBV X GENE TRANSGENIC MICE. Hiroshige Kojima, Kelly D Kaita, Univ of Manitoba, Winnipeg, MB Canada; Z h e n m i n g Xu, James H On, Univ of Southern California, Los Angeles, CA; Yuewen Gong, Gerald Y Minuk, Univ of Manitoba, Winnipeg, MB Canada
ACTIVATION OF NF-KB IN NON-PARENCHYMAL LIVER CELLS AFTER PARTIAL HEPATECTOMY IN RAT: POSSIBLE INVOLVEMENT IN HBEGF EXPRESSION. Shigern Sakuda, Sinji Tamura, Akira Yamada, Jun-lchiro Miyagawa, Koji Yamamoto, Shinichi Kiso, N o b u y u k i ho, Shigeki Higashiyama, Naoyuki Taniguti, Sumio Kawata, Yuji Matsuzawa, Grad Sch of Medicine, Osaka Univ, Suita J a p a n
Background: Hepatocellular carcinoma (HCC) is a c o m m o n t u m o r in patients with chronic hepatitis B virus (HBV) infections. Transgenic mice that express the HBV X protein (HBx) have previously been s h o w n to be more sensitive to the effects of hepatocarcinogens, a l t h o u g h the m e c h a n i s m for this finding remains to be determined. Telomerase activity, the enzyme responsible for maintaining telomere length a n d t h e r e b y c h r o m o s o m a l stability, is absent in most tissues. However, very h i g h levels h a v e been detected in various malignancies including HCC. In rodents, hepatic telomerase activity increases in regenerating livers (Tsujiuchi et al., Cancer Lett. 122: 115-120, 1998). This increase m a y serve to protect hepatocyte DNA from c h r o m o s o m a l instability. In the present study, we hypothesized that the expression of HBx m i g h t interfere with increases in telomerase activity a n d thereby, render these cells more susceptible to carcinogenic mutations. Methods: Eight-week-old CD-1 male mice were obtained from Charles River Laboratories. Two-thirds partial hepatectomies (PHx) were performed by the m e t h o d of Higgins a n d Anderson u n d e r ether anesthesia after fasting for 12 hours. Groups of four to six mice were killed at 0, 6, 12, 24, 36, 48 h o u r s a n d their livers removed a n d snap frozen in liquid nitrogen. Telomerase activity was semi-quantitatively measured by Telomerase PCR ELISA PLus (Roche Diagnostics). F o u r HBx transgenic mice were killed at 12 h o u r s post-PHx w h e n m a x i m u m telomerase activity was observed in the CD-1 non-transgenic mice. mRNA of the telomerase catalytic subunit; mice telomerase reverse transcriptase (mTERT), was q u a n titatively measured by real-time RT-PCR with LightCycler (Roche Diagnostics). Results: Telomerase activity peaked at 12 h o u r s post-PHx in normal mice (baseline; 1.00 ± 0.23 versus 12 h post-PHx; 1.68 - 0.49, p < 0 . 0 5 ) . However, in HBx transgenic mice, telomerase activity was significantly lower, both at baseline (0.71 ± 0.08, p < 0 . 0 5 ) a n d 12 h post-PHx (0.81 -+ 0.09, p < 0 . 0 1 ) . Following PHx, mTERT mRNA expression remained constant in normal mice. In HBx transgenic mice, c o m p a r e d to baseline, mTERT mRNA expression was significantly decreased at 12 h post-PHx (baseline; 2.69 -+ 0.85 versus 12 h; 0.65 -+ 0.29 X 106 copies//zg, p < 0 . 0 1 ) . Conclusion: Hepatic telomerase activity increases following PH in n o r m a l mice. However, in HBx transgenic mice, the increase in telomerase activity is markedly attenuated as is mTERT mRNA expression. These results suggest that HBx expression m a y play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.
Aims: Several transcription factors have been implicated in the initiation of liver g r o w t h after PH. In particular, nuclear factor (NF) KB is activated very rapidly after PH a n d this activation coincides with the transcriptional induction of immediate-earfy g r o w t h response genes. However, the issue of specificafly where NF-KB is activated is unclear. W e previously reported that heparinbinding epidermal growth factor-like g r o w t h factor (HB-EGF) is a hepatotrophic factor. The aims of this s t u d y were to identify NF-KB-activated cells a n d clarify their involvement in HB-EGF expression after PH. Methods: Using rats, a two-thirds PH was performed, after w h i c h NF-KB-activated ceils a n d HB-EGF-positive cells were identified b y Southwestern histochemistry or immunohistochemistry. NF-KB binding activity a n d HB-EGF gene expression in rat hepatocytes a n d n o n - p a r e n c h y m a l cells (NPC) in primary culture were analyzed by electrophoretic mobility shift assay (EMSA) a n d reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results: Southwestern histochemistry for NF-KB a n d i m m u n o h i s t o c h e m i s t r y showed that the first activated-NF-KB-positive cells were Kupffer cells a n d sinusoidal endothelial cells, w h i c h were detectable at 0.5 h after PH. The hepatocyte nuclei became positive for NF-KB a n d the n u m b e r of activated-NF-KB-positive cells reached a m a x i m u m at 1 h after PH. HB-EGF immunoreactivity became to be detected predominantly in these sinusoidal ceils of the periportal zones at 4.5 h after PH, a n d the localization then extended to all lobules at 12 h after PH. HB-EGF gene expression was accompanied by an increase in NF-KB binding activity in NPC in primary culture, w h i c h was abolished b y pyrrolidine dithiocarbamate (PDTC), a n inhibitor of NF-KB activation. Conclusions: NF-KB was activated in NPC prior to activation in hepatocytes. NF-KB activation m a y be involved in HB-EGF expression in N P C after PH.
877
878
DELAYED LIVER REGENERATION W I T H NEGATIVE REGULATION OF HGF AND POSITIVE REGULATION OF TGF-181 MRNA AFTER PORTAL BRANCH LIGATION IN BILIARY OBSTRUCTED RAT. Toshihisa Yamano, Ryuji Hirai, Shinji Hato, Tadahiro Uemura, Nobuyoshi Shimizu, O k a y a m a Univ Sch of Medicine, O k a y a m a J a p a n
PKR LEVELS ARE INCREASED IN HEPATITIS C VIRUS-RELATED HEPATOCELLULAR CARCINOMA. Yoichi Hiasa, Yoshitaka Kamegaya, Ana M Contreras, W e n p i n g He, Massachusetts General Hospital a n d Harvard Medical School, Boston, MA; Emmett V Schmidt, Massachusetts General Hospital Cancer Center, Boston, MA; R a y m o n d T Chung, Massachusetts General Hospital a n d Harvard Medical School, Boston, MA
Introduction: Extended hepateetomy occasionallycarries with it severe post operative liver failure with high morbidity and mortality rates. Preoperative portal branch embolization, which is a modification of portal branch ligation (PBL), induces hypertrophy of remaining non-emhofized hepatic lobe and atrophy ofembolized hepatic lobe to be reseeted. Therefore, this procedure makes extended hepatectomy safer and minimizes postoperative liver dysfunction. However,obstructive jaundice, which may affect the hepatocyte proliferation, has been observed preoperativelyin some cases, such as with hilar cholangiocarcinoma.Biliaryobstruction is known to induce liver fibrosis in association with the up-regulation of intrahepatic transforming growth factor bets (TGF-]3). TGF-~3inhibits the proliferation of hepatocytes, and exerts stimulatory effects on fibrogenesisby activating hepatic stenate cells (HSCs). Activated HSCs produce a large amount o[ TGF-~ and express the alpha-smooth muscle actin (a-SMA) isoform. On the other hand, hepatocyte growth factor (HGF) is produced chiefly from HSCsand acts as the most potent mitogen on hepatoeytes. However, HGF mRNA expression is lost once HSCs transform into myofibroblast-likecells. Although several studies regarding the liver regeneration after partial hepatectomy or PBL with obstructivejaundice have been performed, it is not clear how the obstructive jaundice affects the liver regeneration after those treatment. Moreover, there were few studies regarding the expression of growth factors in the regenerating liver after those treatments with obstructivejaundice. In this study, we evaluated the effect of obstructive jaundice on liver regeneration after PBL, and the expression of HGF and TGF-181mRNAafter PBLin jaundiced rat liver. Methods: MaleWistsr rats were used. In BDPVLgroup, common bile duct was Iigated,and 7 days later (Day0) the left branch of portal vein, feeding left median and lateral lobes, was ligated. In PVL group, common bile duct was exposed, but not ligated, and 7 days later (Day 0), rats underwent only PBL.After PBL,rats were sacrificed at various times. Liver wet weight, proliferating cell nuclear antigen (PCNA) labeling, HGF and TGF-]31 mRNA expression, and immunohistochemicaI staining with ~-SMA antibody were studied. Results: In the BDPVL and PVL groups, the wet weight ratio of the non-ligated lobe to the whole liver increased prompdy after Day 0. In the PVLgroup, the ratio on Day 5 increased to 81-+1% and was significantly higher than the BDPVLgroup ratio (58_+3%). However,by Day 10, the ratio in BDPVLgroup increased to 79_+3%,close to the PVLgroup ratio (83±3%) at that time. The PCNA labeling index in the non-ligated lobe significantly increased after Day 0 and reached a peak at Day I in both the BDPVLand the PVL groups. The peak level, however, was significantlylower in the BDPVLgroup (19 ~ 1%) than in the pVL group (31±4%). The expression of HGF mRNAremarkably increased after Day 0 in the non-figated growing lobe and the peak was observed at 12 hours in both groups. However,the peak levelin the BDPVLgroup was significantlylower than in the PVL group. The expression of TGF-,81also increased between Day 2 and 5 in the non-ligated lobe of the BDPVLand PVL groups, and the level in the BDPVL group was significantlyhigher than in the PVLgroup, a-SMAstaining showed that hepatic stellate cells (HSCs) were gradually activated into myofibroblast-likecells in the BDPVLgroup. Conclusion: This study has shown that obstructivejaundice has a strong effect on the expression of HGF and TGFq81 mRNA,which are regulated in the negative direction for liver regeneration, resulting in delayed liver regeneration. Furthermore, this study has shown that bfliary obstruction activates HSCs. These findings suggest that HSCs may play an important role in the regulation of the expression of HGF and TGF-/31mRNAin the jaundiced rat liver.
Background/Aims: Hepatitis C virus (HCV) is a leading cause of hepatocellular carcinoma (HCC) worldwide. The double-stranded RNA-activated protein kinase (PKR) mediates the phosphorylation of eukaryotic translation initiation factor elF2-a, w h i c h m a y in turn inhibit HCV protein synthesis. In addition to its putative effects o n viral protein synthesis, PKR also inhibits host cell growth a n d proliferation a n d has therefore been p r o p o s e d to act as a t u m o r suppressor. Recent reports that the HCV NS5A protein tightly binds a n d inhibits PKR activity 1 a n d that HCV E2 inhibits PKR kinase function 2 raise the possibility that HCV contributes to HCC t h r o u g h alterations in PKR. In order to clarify the m e c h a n i s m of HCV-related hepatocarcinogenesis, we examined the expression of PKR in paired malignant a n d non-malignant tissue from patients with HCV-related HCC. In addition, we also sought to determine whether mutations in PKR sequences could be identified from these tissues. Methods: We prepared two sets of primers flanking the PKR E2- a n d the PKR NS5Abinding sites, respectively. Tissue samples were obtained from 9 HCV-infected patients with cirrhosis a n d H C C w h o had u n d e r g o n e liver transplantation or resection. T u m o r (T) a n d s u r r o u n d i n g n o n t u m o r o u s (NT) cirrhotic tissue was extracted for RNA a n d protein. W e performed semiqnantitative RT-PCR using GAPDH as a housekeeping control to determine the relative expression of PKR mRNA between T a n d NT tissue. In addition, TOPO cloning was performed on each cDNA amplicon a n d individual transformants were sequenced (5-10 clones per amplicon). Western blot was performed using pofyclonal antisera for PKR. Results: W e found no significant differences in levels of PKR mRNA between T and NT pairs for either the E2- or NS5A-binding regions. W h e n sequences of individual clones corresponding to each mRNA amplicon were analyzed, we found no mutations in either T or NT tissue. However, PKR protein levels were significantly increased in T compared to NT in all 9 pairs (range 1.2-4.3 fold, mean 2.6, p = 0 . 0 0 0 1 ) . Conclusion: While there are no significant differences in PKR gene expression or sequences between T a n d NT tissue in HCV-related HCC, levels of PKR protein are significantly elevated in HCC tissue. These data suggest that PKR expression is altered at the posttranscriptional level in h u m a n HCC. The finding of increased levels of PKR protein in malignant tissue raises the possibility that PKR does not function as a classical t u m o r suppressor protein driving the formation of HCC. Reference: ZGale M Jr., et al Virology 230 217, 1997.2Taylor DR, et al. Science 285: 107, 1999.