Retrospective analysis of in vivo recovery and clearance during continuous infusion of recombinant factor VIII products: a single-institution study.

BACKGROUND: Continuous infusion (CI) of recombinant FVIII (rFVIII) concentrates has been reported as an effective and safe method to achieve haemostasis during major surgeries or severe bleeding events. For more effective and safer CI, better understanding of in vivo recovery (IVR) and clearance (CL) issues is imperative. OBJECTIVE: We investigated the following factors affecting IVR and CL using univariate and multivariate regression analyses during 47 CIs in 34 patients: rFVIII concentrate type, haemophilia severity, blood type, the presence of hepatitis C virus (HCV) or human immunodeficiency virus (HIV), age and body mass index (BMI). RESULTS: The mean IVR was 1.64 ± 0.49 IU dL-1 per IU kg-1 , and the mean CL during CI was 3.56 ± 1.57 mL h-1 kg-1 . The univariate and multivariate regression analyses showed that the CL of octocog alfa was significantly lower than that of rurioctocog alfa (P = 0.043 and 0.0034, respectively). There was a significant difference in BMI in the univariate and multivariate regression analyses (P = 0.0403 and 0.0376, respectively). CONCLUSIONS: This study indicated that CL during CI was potentially affected by the type of rFVIII concentrate used and BMI.

Epicatechin downregulates adipose tissue CCL19 expression and thereby ameliorates diet-induced obesity and insulin resistance.

BACKGROUND AND AIMS: Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. METHODS AND RESULTS: DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (CC motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. CONCLUSIONS: EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.

Intraoperative portal venous pressure and long-term outcome after curative resection for hepatocellular carcinoma.

BACKGROUND: Outcomes of liver resection for hepatocellular carcinoma (HCC) have improved owing to better surgical techniques and patient selection. Portal hypertension may influence outcome but the preoperative definition and role of portal hypertension are far from clear. The aim of this study was to elucidate the influence of portal venous pressure (PVP) measured directly during surgery on outcomes of liver resection in patients with HCC. METHODS: Patients who had resection of HCC between 1997 and 2009, and who underwent direct measurement of PVP immediately after laparotomy were enrolled. These patients were divided into groups with high (at least 20 cmH(2)O) and low (less than 20 cmH(2)O) PVP. The influence of PVP on overall and recurrence-free survival was analysed and prognostic factors were identified. RESULTS: A total of 177 patients were enrolled, 129 in the low-PVP group and 48 in the high-PVP group. The 5-year overall survival rate (63·7 versus 31 per cent; P < 0·001) and recurrence-free survival rate (52·5 versus 12 per cent; P < 0·001) were significantly higher in patients with low PVP. In multivariable analysis, two or more tumours, tumour diameter at least 5 cm, high PVP, grade B liver damage and Hepatic Activity Index (HAI) grade 7 or more were significant predictors of poorer survival after liver resection. Two or more tumours, tumour diameter at least 5 cm and HAI grade 7 or more were significant predictors of poorer recurrence-free survival. CONCLUSION: High PVP was associated with poor long-term outcome after liver resection for HCC.

Is liver-targeted FOXp3 staining beneficial after living-donor liver transplantation?

As treatments for acute cellular rejection (ACR) and recurrent hepatitis caused by hepatitis C virus (HCV) are dramatically different, making a precise diagnosis is considered to be essential in patients after liver transplantation. Therefore, we investigated whether immunohistochemical detection of FOXp3, a marker for regulatory T cells (CD4+ CD25+), could be used to differentiate between recurrent hepatitis C and ACR. From a group of 103 cases of living-donor liver transplantation (LDLT), 48 samples were taken via liver biopsy from 20 patients with HCV infection. An initial diagnosis was made based on hematoxylin and eosin staining, which was scored with the hepatitis activity index (HAI) grading, whereas ARC was scored with the rejection activity index (RAI). The FOXp3 immunohistochemical staining on serial specimens was retrospectively analyzed, scoring from 0 to III. The time after LDLT was a median of 270 (range: 14-2000) days, whereas the median number of biopsies per patient was 3 (range: 1-8). The HAI was significantly different between 0 vs. I, and II vs. III, in terms of the FOXp3 score. On the other hand, a significant difference in the RAI was only found between 0 vs. I. In conclusion, FOXp3 may represent a surrogate marker for recurrent HCV infection after LDLT.

Recurrence-free survival more than 10 years after liver resection for hepatocellular carcinoma.

BACKGROUND: High recurrence rates after liver resection with curative intent for hepatocellular carcinoma (HCC) remain a problem. The characterization of long-term survivors without recurrence after liver resection may help improve the therapeutic strategy for HCC. METHODS: A nationwide Japanese database was used to analyse 20 811 patients with HCC who underwent liver resection with curative intent. RESULTS: The 10-year recurrence-free survival rate after liver resection for HCC with curative intent was 22.4 per cent. Some 281 patients were recurrence-free after more than 10 years. The HCCs measured less than 5 cm in 83.2 per cent, a single lesion was present in 91.7 per cent, and a simple nodular macroscopic appearance was found in 73.3 per cent of these patients; histologically, most HCCs showed no vascular invasion or intrahepatic metastases. Multivariable analysis revealed tumour differentiation as the strongest predictor of death from recurrent HCC within 5 years. CONCLUSION: Long-term recurrence-free survival is possible after liver resection for HCC, particularly in patients with a single lesion measuring less than 5 cm with a simple nodular appearance and low tumour marker levels.

Helicobacter bilis colonization of the biliary system in patients with pancreaticobiliary maljunction.

BACKGROUND: Helicobacter bilis is considered to be a causative factor in the pathogenesis of biliary cancer. This study investigated the prevalence of H. bilis colonization of the biliary system of patients with pancreaticobiliary maljunction (PBM). METHODS: Bile juice and biliary tissue samples were collected from 17 patients with PBM and 27 controls who had benign biliary disease without PBM. DNA extracted from each biliary sample was subjected to polymerase chain reaction (PCR) analysis for H. bilis and Helicobacter pylori. RESULTS: PCR assays revealed that 12 of the 17 patients with PBM were positive for H. bilis DNA, compared with eight of 27 patients without PBM (P = 0.009). Among patients with PBM, H. bilis DNA was identified in six of eight children, including a 2-month-old infant, and in six of nine adults. The high prevalence of H. bilis DNA in the biliary system of patients with PBM was independent of age, sex, common bile duct dilatation, configuration of the pancreatic and bile ducts, and amylase activity in bile. CONCLUSION: H. bilis colonization of the biliary system is extremely common in patients with PBM. This may point to a role in the pathogenesis of biliary cancer.

Cerebellar ataxia in a patient receiving calcineurin inhibitors after living donor liver transplantation: a case report.

Neurological complications of calcineurin inhibitors are frequent problems after transplantation. Cerebellar ataxia with other neurological findings and an abnormal density area in the subcortical white matter are found by MRI in the brains of most patients with central nervous system complications caused by calcineurin inhibitors. Such neurological complications are not life-threatening, but have a negative impact on the quality of life. We describe a 58-year-old woman who developed cerebellar ataxia at 4 days after living donor liver transplantation. She walked with a swaying gait, and after walking for 5 minutes she was unable to stand. Her symptoms persisted after a change from tacrolimus to cyclosporine, but dose reduction of cyclosporine and addition of mycophenolate mofetil cured the ataxia. We diagnosed a case of cerebellar ataxia without leukoencephalopathy or other neurological symptoms, as a new complication of calcineurin inhibitor treatment. We concluded that careful attention should be paid to neurological complications of calcineurin inhibitors.

Role of carcinoembryonic antigen in the progression of colon cancer cells that express carbohydrate antigen.

Carcinoembryonic antigen (CEA) has been reported to promote the metastatic potential in some experimental tumors. Adhesion molecules are known to play an important role in the process of metastasis. Cytokines, including interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are produced by Kupffer cells, induce endothelial cells to express adhesion molecules. As a result, the present study was designed to investigate whether the interaction between CEA and Kupffer cells accelerated the metastatic potential of tumors in the liver. Kupffer cells isolated from the liver of male BALB/c mice were cultured with CEA, either with or without the addition of a cytokine inhibitor. The levels of IL-1beta and TNF-alpha were examined in a culture medium. An adhesion assay of colon cancer cell lines to human umbilical vein endothelial cells was also performed. When CEA was added to the Kupffer cell culture medium, cytokines were produced. Elevated levels of cytokines appeared to lead to increased rates of adhesion of cancer cells to endothelial cells. However, these phenomena were blocked by the addition of cytokine inhibitors. CEA stimulated Kupffer cells to produce cytokines. An elevated number of cytokines have been proven to promote the expression of adhesion molecules in endothelial cells. These processes are therefore considered to contribute to the metastasis of malignant cells to the liver. These results suggest that cytokine inhibitors may therefore play an important role in the inhibition of hepatic metastasis.

Drug-induced down-regulation of topoisomerase I in human epidermoid cancer cells resistant to saintopin and camptothecins.

The anticancer agent saintopin induces DNA cleavage mediated by both topoisomerase (topo) I and topo II in vitro through stabilization of the reversible enzyme-DNA cleavable complex. We established saintopin-resistant cell lines (KB/STP-1 and KB/STP-2) from human epidermoid cancer KB cells by stepwise exposure to increasing doses of the drug. KB/STP-1 and KB/STP-2 cells showed 12- and 44-fold increases, respectively, in resistance to saintopin relative to that of KB cells. Both saintopin-resistant cell lines showed only small reductions in sensitivity to the topo II inhibitor etoposide but developed marked cross-resistance to the topo I-targeting camptothecin derivative CPT-11 [(4s)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbony loxy] dione hydrochloride trihydrate] and its active form, SN-38 (7-ethyl-10-hydroxycamptothecin). In contrast, both KB/STP-1 and KB/STP-2 cells showed increased collateral sensitivity to cisplatin, a nitrosourea derivative, mitomycin C, and UV light. The protein concentration, activity, and mRNA abundance of both topo I and topo II were similar in KB/STP-1, KB/STP-2, and the parental KB cells. There were no significant changes in the drug-stabilized topo-DNA cleavable complex formation in KB and KB/STP-2 cells. Two point mutations were detected in topo I cDNA from KB/STP-2 cells, but these were also present in KB cells. Topo I mRNA abundance decreased markedly immediately after exposure of KB/STP-2 cells to saintopin; no such effects were apparent in KB cells. In contrast, topo II mRNA was not markedly affected by saintopin in either KB or KB/STP-2 cells. Treatment with CPT-11 or SN-38 also induced a markedly greater and more persistent reduction in topo I mRNA abundance in KB/STP-2 cells than in KB cells. Etoposide had no marked effect on topo I mRNA abundance in either KB/STP-2 or KB cells. Topo I mRNA was highly unstable in KB/STP-2 cells in comparison to KB cells when incubated with saintopin. This novel regulation of topo I mRNA by topo I-targeting agents could be associated with acquirement of drug resistance to saintopin or SN-38/CPT-11 in KB/STP-2 cells.

Temporary interruption of regional blood flow combined with local hyperthermia for cancer chemotherapy.

A striking chemotherapeutically curative effect on tumor was obtained by means of temporary interruption of regional blood flow combined with local hyperthermia. By analyzing various basic conditions required for this system using Ehrlich tumor implanted in the hind limbs of mice, the following were found to be essentially indispensable to obtain satisfactory chemotherapeutic effects: (a) a time interval of 1 to 3 min after systemic i.v. administration of drug to the mice, (b) use of a tourniquet on the tumor-bearing mouse limb to stop blood flow, and (c) warming at 37-41 degrees (d) for a period of at least 30 to 60 min. Among the chemotherapeutic drugs tested in the present study, Carbazilquinone (NSC 134679) was the most effective because it revealed the strongest antitumor effect despite its relative innocuousness to nontumorous adjacent normal tissues. Applying the present method, a large syngeneic mouse sarcoma transplanted to the limb 7 days before the experiment also completely regressed in 6 of 9 mice.

Hepatitis C and hepatitis B in the etiology of hepatocellular carcinoma in the Japanese population.

We conducted case-control studies of hepatocellular carcinoma (HCC) and liver cirrhosis (LC) in relation to hepatitis C virus (HCV) and hepatitis B virus infection, involving 91 patients with HCC, 75 patients with LC who had no evidence of HCC, and 410 control subjects from the Japanese population. Serum antibody to HCV (anti-HCV) was detected by both enzyme-linked immunosorbent assay and recombinant immunoblot assay in 51, 51, and 3% of HCC, LC, and controls, respectively, whereas the corresponding prevalence of serum hepatitis B surface antigen (HBsAg) was 21, 11, and 2%, respectively. The relative risks (and 95% confidence intervals) for the presence of serum anti-HCV were estimated as 52.3 (23.9-114.3) for HCC and 64.4 (27.4-151.4) for LC. These values exceeded the relative risk of HCC (15.3) and that of LC (6.1) for positive serum HBsAg. Among male patients with HCC or LC, anti-HCV rates were very high in blood recipients (about 70%), heavy drinkers (46-62%), and those who had no identifiable risk factors (65-75%), indicating possible transmission of HCV via routes other than transfusion. No significant difference in anti-HCV status was observed between the HCC and LC groups. It was notable that anti-HCV was much less prevalent among HBsAg-positive patients with HCC or LC than among HBsAg-negative ones. There was a slight to moderate increase in HCC or LC risk among blood recipients and heavy drinkers after adjustment for anti-HCV status. These results indicate that, in Japan, the possible role of HCV infection in the etiology of HCC and LC is extremely large and seems to be more important than chronic hepatitis B virus infection.

D-myo-inositol 1,4,5-trisphosphate analogues substituted at the 3-hydroxyl group.

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogues derived at 3-OH with a bulky substituent were chemically synthesized and structural features of vicinity surrounding the 3-OH of Ins(1,4,5)P3, recognized by metabolic enzymes and by the receptor were explored. 3-Benzoyl-, 3-methylbenzoyl- and 3-para-aminobenzoyl-Ins(1,4,5)P3 inhibited the dephosphorylation of [3H]Ins(1,4,5)P3 by the 5-phosphatase present in erythrocyte ghosts, but the potency varied. The inhibitory potency for the former two compounds was slightly lower than that for Ins(1,4,5)P3, while that for the latter compound was higher. Transfer of the amino group to the meta-position of the benzoyl group led to a less potent analogue. In an assay of [3H]Ins(1,4,5)P3 3-kinase at a low Ca2+ concentration, catalyzed by rat brain cytosol, 3-meta-aminobenzoyl-Ins(1,4,5)P3 was the most potent among compounds examined, including Ins(1,4,5)P3 in inhibiting the phosphorylation, whereas both 3-benzoyl- and 3-methylbenzoyl-Ins(1,4,5)P3 at concentrations up to 30 microM, were without effect. All analogues examined were effective in inhibiting [3H]Ins(1,4,5)P3 binding to purified Ins(1,4,5)P3 receptor, but all 3-derived analogues were less potent and 3-benzoyl-Ins(1,4,5)P3 was the least potent. It would thus appear that the space in the vicinity surrounding the 3-hydroxyl group of Ins(1,4,5)P3 is sterically restrictive with regard to recognition by metabolic enzymes and the receptor, whereas the amino group providing arms for either the electrostatic interaction or the hydrogen bond, makes the analogues more potent.

Synthetic inositol 1,3,4,5-tetrakisphosphate analogs and their effect on the binding to microsomal fraction of rat cerebellum.

Binding activity of [3H]inositol 1,3,4,5-tetrakisphosphate (InsP4) was characterized with rat cerebellar membranes. Two types of InsP4 analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP4 have been synthesized and their effects on the binding activity were also examined. [3H]InsP4 binding was gradually displaced by increasing amounts of unlabeled InsP4, with an IC50 of 60-170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca2+, this being in clear contrast with [3H]InsP3 binding noted in the same species of tissue. Heparin inhibited the binding, with an IC50 of 1.7, 3 or 20 micrograms/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [3H]InsP3 binding. InsP4 analogs were as effective as InsP4 in displacing [3H]InsP4 from rat cerebellar membranes, thereby indicating that the 2nd hydroxyl group may not be involved in recognition of InsP4 by its binding sites.

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Frequency of apoptosis of tumor-infiltrating lymphocytes induced by fas counterattack in human colorectal carcinoma and its correlation with prognosis.

We investigated apoptosis in tumor-infiltrating lymphocytes (TILs) obtained from 41 colorectal carcinomas by in situ nick translation (ISNT). When the ISNT labeling index (LI) was determined as the number of positive nuclei per 1000 nuclei of TIL in tissue sections, the median LI was 12.0 (range, 2-30). The ISNT LI of colorectal carcinoma with lymph node metastasis was higher than that of colorectal carcinoma without metastasis. The cases with a high LI of 212.0 had a significantly poorer prognosis than those with a low LI. We also confirmed immunohistochemically that a part of the TILs expressed Fas using the sections adjacent to what contained abundant ISNT-positive TILs. Moreover, Fas ligand (FasL) expression was detected on the cell surface as well as the cytoplasm of colorectal cancer cells in 61% of cases. Apoptosis in TILs was consistently seen more frequently in FasL-positive cases than in FasL-negative ones. These findings indicate that the FasL expressed in colorectal carcinoma cells may kill the Fas-positive immune effective TILs by means of a Fas-FasL system termed Fas counterattack. This tumor immune evasion induced by FasL may therefore affect the malignant potential of human colorectal carcinoma.

Experimental study on pathogenesis and histomorphology of early carcinoma of the extrahepatic bile duct in the Syrian hamster.

To elucidate the pathogenesis of carcinomas in the extrahepatic bile duct, we investigated the histomorphological characteristics of adenomas and early carcinomas induced in the extrahepatic bile duct of hamsters. Syrian hamsters underwent a cholecystoduodenostomy along with a dissection of the common duct, while also being administered N-nitrosobis(2-oxopropyl)amine (BOP). The tumors that arose from the extrahepatic bile duct included 10 adenomas and 55 early carcinomas in 56 of the 156 hamsters sacrificed. All the adenomas were found to be polypoid in shape. The early carcinomas, which were restricted within the mucosal layer of the bile duct, showed the following three different growth patterns: (1) protruding type in 41 (75%), consisting of 27 polypoid and 14 papillary tumors; (2) superficial spreading type in 9 (16%); and (3) periductal glandular type in 5 (9%). There were no depressed tumors observed. Carcinomas existing either alone or associated with adenomas were evident in 12 (22%) tumors, and 11 of these were polypoid. Atypical papillary hyperplasia within the tumor mass was noted in 22 early carcinomas (40%) and was particularly prominent in papillary type tumors. These results support the concept of an adenoma-carcinoma sequence in the majority of polypoid tumors of the extrahepatic bile duct. Atypical papillary hyperplasia might also be premalignant, and these precursor lesions should reflect the growth patterns of tumors, at least in the early stage of tumorigenesis.

Inhibitory effects of safe and novel SOD derivatives, galactosylated-SOD, on hepatic warm ischemia/reperfusion injury in pigs.

BACKGROUND/AIMS: Oxygen-derived free radicals such as superoxide play an important role in ischemia/reperfusion (IR) injury during and after extensive liver surgery or liver transplantation. Superoxide dismutase (SOD) has protective effects against hepatic IR injury. The effect of native SOD is, however, limited because of rapid elimination from the blood circulation and poor affinity for liver cells. It was reported by our collaborators that a SOD derivative modified with galactose (Gal-SOD) was selectively delivered well to hepatocytes by direct attachment to galactose receptors. In the present study, the efficacy of this agent for attenuating hepatic warm IR injury was investigated using the pig model. METHODOLOGY: After 45-min clamping of the hepatic artery and portal vein, pigs were divided into 3 groups according to the following treatments. Ten milliliters of normal saline in Group 1 (n=5), 10,000 units/kg of native SOD in Group 2 (n=5) and 10,000 units/kg of Gal-SOD in Group 3 (n=5) were given just prior to hepatic reperfusion. Liver function including clearance of total bile acid (TBA) and hyaluronic acid (HA) was investigated. Lipid peroxidase of the liver tissue (LPO) and histological findings were examined. In addition, survival rates of the pigs in each group were evaluated. RESULTS: The survival rates at the 7th day after the operation were 60%, 80%, 100% in Groups 1, 2 and 3, respectively. Liver function tests, clearance of TBA and HA, and LPO levels were significantly improved in Groups 3 over findings in Groups 1 and 2. Congestion of hepatic tissues and vacuolization of hepatocytes in Group 3 were less than those in Groups 1 and 2. These results suggested that oxygen-derived free radicals were scavenged by Gal-SOD and IR injury was attenuated. CONCLUSIONS: A safe and novel agent, Gal-SOD has a protective effect against hepatic warm IR injury.

Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation.

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunction with radiation therapy using an HVJ-liposome vector system. The radio-inducible suicide gene was constructed by insertion of the early growth response gene 1 (Egr-1) promoter upstream of the HSV-tk gene (EGF-tk). First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry were performed. The Egr-1 gene was highly expressed in HCC compared with normal liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepatocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by luciferase assay as a reporter gene system. Egr-1 promoter activity was markedly increased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, only a twofold increase in activation was noted in normal hepatocytes. An in vitro gene therapy experiment showed that EGR-tk-transduced hepatoma cells became highly sensitive to ganciclovir (GCV) after irradiation, but not without irradiation. GCV with or without irradiation did not show any cytotoxic effects against control gene-transfected cells. In addition, a "radiosensitization effect" was also demonstrated by combination therapy with the HSV-tk/GCV system and irradiation. To examine the efficacy of this EGR-tk/GCV gene therapy in vivo, xenografted liver tumors in nude mice were targeted using the HVJ-liposome vector system. EGR-tk-transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In comparison, tumors continued to grow in all mice (n = 8 in each group) treated by transfer of EGR-tk followed by either irradiation without GCV or GCV without irradiation. Our data indicate that HSV-tk gene therapy under the control of a radioinducible promoter is effective, and might be selective for hepatoma cells because of its inducible and radiosensitive capacity after radiation exposure as well as its tumor-specific activation.