Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats.

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II) has been suggested to play an important role in liver fibrogenesis. It induces hepatic stellate cell (HSC) proliferation and up-regulates the transforming growth factor beta(1) (TGF-beta(1)) expression via AT-II type 1 receptor (AT(1)-R) in vitro. The aim of the present study was to examine the in vivo effect of candesartan (CA), a clinically used AT(1)-R blocker (ARB), and perindopril (PE), an angiotensin-converting enzyme (ACE) inhibitor (ACE-I), on pig serum-induced liver fibrosis development in rats. The clinically available comparable doses of CA and PE significantly attenuated the fibrosis development. These inhibitory effects of PE and CA were also found in the on-going liver fibrosis model. The hepatic hydroxyproline and serum fibrosis markers were significantly suppressed by CA and PE treatment. Furthermore, the alpha smooth muscle actin (alpha-SMA) positive cells in number were markedly suppressed by CA and PE treatment. Similarly, the hepatic TGF-beta(1) protein and messenger RNA (mRNA) levels were significantly suppressed. Our in vitro study showed that AT-II increased the TGF-beta(1) mRNA expression in the activated HSCs, and this effect was totally blocked by CA. These results suggested that the RAS, especially AT-II and AT(1)-R interaction plays a pivotal role in liver fibrosis development through HSC activation. Because both CA and PE are widely used in clinical practice without serious side effects, these drugs may provide an effective new strategy for anti-liver fibrosis therapy.

The vascular endothelial growth factor receptor KDR/Flk-1 is a major regulator of malignant ascites formation in the mouse hepatocellular carcinoma model.

The vascular endothelial growth factor-A (VEGF-A), also known as the vascular permeability factor (VPF), has been shown to play an important role in malignant ascites formation. The effects of VEGF-A are mediated through flt-1 and kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1) receptors. It has been shown that KDR/Flk-1 is a predominant receptor in solid hepatocellular carcinoma (HCC) development, but the role of this receptor in hepatic ascites formation has not yet been elucidated. In this study, we examined the role of KDR/Flk-1 in the murine MH134 hepatic malignant ascites formation by means of VEGF-A- and KDR/Flk-1-specific neutralizing antibodies (VEGF-A nAb and KDR/Flk-1 nAb, respectively). The mean volume of ascites, number of tumor cells in ascites, and the peritoneal capillary permeability were significantly suppressed by VEGF-A nAb and KDR/Flk-1 nAb treatment. These inhibitory effects of KDR/Flk-1 nAb were more potent than those of VEGF-A nAb. The autophosphorylation of KDR/ Flk-1 in the peritoneal wall was almost completely abolished by KDR/ Flk-1 nAb, whereas a certain level of activation was still shown by VEGF-A nAb treatment. Another VEGF-family, VEGF-C, which also binds KDR/Flk-1, was detected in the ascites. Furthermore, in the therapeutic experiment, although both VEGF-A nAb and KDR/Flk-1 nAb prolonged the survival rate of ascites-bearing mice, the latter showed a more significant impact on the survival of animals. These results suggest that KDR/Flk-1 is a major regulator of malignant hepatic ascites formation, and that in addition to VEGF-A, VEGF-C may also be involved in the malignant ascites formation via KDR/ Flk-1 activation.

The angiotensin-I-converting enzyme inhibitor perindopril suppresses tumor growth and angiogenesis: possible role of the vascular endothelial growth factor.

Angiotensin-I converting enzyme (ACE) inhibitor is used widely as an antihypertensive agent, and it has been suggested recently that it decreases the risk of cancer (A. F. Lever et al., Lancet, 352: 179-184, 1998). In this study, we examined the effect of several ACE inhibitors and angiotensin-II type 1 receptor (AT(1)-R) antagonists on tumor development and angiogenesis in a murine hepatocellular carcinoma model. Among ACE inhibitors, perindopril appeared to be a potent inhibitor of tumor development and angiogenesis, whereas AT(1)-R antagonists did not exert such an inhibitory effect. The inhibitory effect of perindopril was achieved even on established tumors. The level of the potent angiogenic factor, vascular endothelial growth factor (VEGF), in the tumor was significantly suppressed by perindopril. In vitro studies showed that perindopril-derived active form, perindoprilat, suppressed the endothelial cell tubule formation. Perindoprilat treatment also significantly inhibited VEGF mRNA expression in BNL-HCC cells in vitro. These results showed that the ACE inhibitor perindopril inhibited tumor development and angiogenesis independent from AT(1)-R blockage, and that VEGF alternation may be involved in the mechanism of this inhibitory effect. Because perindopril is widely used in clinical practice, it may represent an effective new strategy for anticancer therapy.

A potential approach for electrochemotherapy against colorectal carcinoma using a clinically available alternating current system with bipolar snare in a mouse model.

BACKGROUND: Although electrochemotherapy appears promising for the treatment of superficial tumors, its usefulness against internal tumors, such as colorectal carcinoma (CRC), has not been well examined. Furthermore, since direct current electric pulses have been used for electropermeabilization of tumors in all in vivo electrochemotherapy studies, including clinical trials, the usefulness of alternating current systems has not been examined at all. In a mouse model it was examined whether the alternating current system with a bipolar snare, which has been employed already as a clinical endoscopic treatment modality, was useful for electrochemotherapy against CRC. METHODS: Murine CRC colon 26 cells were implanted subcutaneously into syngeneic BALB/c mice and electrochemotherapy with bleomycin (BLM) using the alternating current system was performed against established CRC tumors. RESULTS: Electrochemotherapy significantly suppressed the growth of established CRC tumors, resulting in significantly prolonged survival of animals with CRC. Histological examination revealed that electrochemotherapy caused massive necrosis of CRC tumors. Subsequent analysis revealed that the delivery of alternating current electric pulses to CRC tumors profoundly increased intratumoral amounts of BLM. CONCLUSIONS: Because the alternating current system using a bipolar snare has been used widely as an endoscopic treatment modality in clinical settings, these results indicate that electrochemotherapy using the alternating current system may be a promising approach for the treatment of CRC.

Amelioration of retrovirus-mediated gene transfer into hepatocellular carcinoma cells.

Recombinant retroviruses are by far the most frequently used vehicle in clinical gene therapy. No serious side-effects have been reported so far in clinical gene therapy trials using recombinant retroviral systems. Low titers of recombinant retroviruses, however, have limited the usefulness of recombinant retroviruses. To improve the efficiency of retrovirus-mediated gene transfer, we previously introduced the polyomavirus early region into amphotropic PA317 cells and established a modified retroviral packaging cell line, PAMP51. We demonstrate here that recombinant retroviruses produced by PAMP51-derived retroviral producing cells have approximately 10-fold higher titers compared with those produced by conventional PA317-derived retroviral producing cells. Importantly, recombinant retroviruses produced by PAMP-derived retroviral producing cells could infect hepatocellular carcinoma cells much more efficiently and could induce much stronger expression of a lacZ reporter gene in HCC cells compared with those produced by PA317-derived ones. These results indicate that recombinant retroviruses prepared from PAMP51-derived retroviral producing cells are much more useful than those prepared from PA317-derived ones and that the use of PAMP51 retroviral packaging cells may open up new avenues for the treatment of various types of cancer including hepatocellular carcinoma.

Intravenous readministration of an adenoviral vector performed long after the initial administration failed to induce re-expression of the original transgene in rats.

Although most humans have been exposed to wild-type adenoviruses in their childhood, titers of neutralizing antibodies against viruses decrease with the passage of time. In the present study, we infused adenoviruses carrying the lacZ gene into the tail vein of rats, and re-infused the same adenoviruses long after the initial administration. However, development of neutralizing antibodies against adenovirus and proliferation of adenovirus-specific T cells were elicited profoundly by adenoviral readministration, and transgene expression was not induced in rats. Our results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings for the treatment of cancer.

Adenovirus-mediated gene transfer into tumors: evaluation of direct readministration of an adenoviral vector into subcutaneous tumors of immunocompetent mice.

Because systemic administration of adenoviruses appears to be limited by their immunogenicity, we examined the feasibility of intratumoral administration of adenoviruses. Direct intratumoral administration of adenoviruses resulted in efficient but transient transgene expression. When adenoviruses were readministered directly into tumors, re-expression of the transgene was achieved. Transgene expression induced by the adenoviral readministration was, however, markedly weaker than that induced by the initial administration. Furthermore, intratumoral readministration of adenoviruses elicited profound humoral and cellular immune responses to adenoviruses. These results may have important implications for efficacy considerations when adenoviral vectors are used for clinical cancer gene therapy.

Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/ enhancer. A model of CCl(4)-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(alpha1)-collagen-I, (alpha2)-collagen-IV, and alpha-smooth muscle actin (alpha-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl(4), however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl(4)-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl(4)-treated TIMP-Tg-mice with a pattern similar to that of alpha-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development.

Retrovirus-mediated in vivo gene therapy using the herpes simplex virus thymidine kinase gene against carcinomatous peritonitis.

BACKGROUND: Carcinomatous peritonitis is characterized by massive malignant ascites, while peritoneally disseminated carcinomatosis is characterized by a large number of metastatic solid tumors in the peritoneal cavity. Although both are fatal end-stage manifestations of malignancies derived from the digestive system, the former is usually more serious than the latter due to massive malignant ascites. Although the effectiveness of gene therapy against peritoneally disseminated carcinomatosis has been shown in animal experiments, its effectiveness against carcinomatous peritonitis remains to be examined. METHODS: A carcinomatous peritonitis model was made by inoculating murine hepatocellular carcinoma cells, MH134, into the peritoneal cavity of syngeneic C3H/He mice, resulting in production of massive malignant ascites without development of intraperitoneal solid tumors. Model animals were injected intraperitoneally with retroviruses carrying the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) treatment. RESULTS: Retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system was shown to have a significant impact on survival of animals with carcinomatous peritonitis not only at an early stage, but also at an advanced stage. Furthermore, repeated injections of HSV-tk-carrying retroviruses significantly prolonged the survival of animals with carcinomatous peritonitis compared with a single injection protocol. When intraperitoneal administration of recombinant interleukin-2 (IL-2) was added to the HSV-tk/GCV system, levels of IL-1beta and IL-2 in malignant ascites were significantly increased, resulting in significantly reduced ascite volume and prolonged survival. CONCLUSIONS: Our results indicate the feasibility of retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system plus IL-2 treatment against carcinomatous peritonitis.

Electrochemotherapy for colorectal cancer with commonly used chemotherapeutic agents in a mouse model.

We examined here the usefulness of electrochemotherapy against colorectal cancer (CRC) using a mouse model. Electropermeabilization profoundly increased the sensitivity of murine CRC, Colon 26, and MC38 cells to bleomycin (BLM) but not to 5-fluorouracil (5-FU) or to cisplatin (CDDP) in vitro. In vivo experiments revealed that electrochemotherapy with 5-FU, CDDP, or BLM was much more effective against CRC compared with the treatment of the drug alone. Electrochemotherapy with BLM or CDDP exhibited profound antitumor effects on subcutaneously established CRC in mice, and complete tumor regression was observed in five and four of eight animals, respectively. Electrochemotherapy with 5-FU also had an impact on CRC development, and complete cure was observed in one of eight animals. Subsequent analyses revealed that electropermeabilization significantly increased intratumoral amounts of BLM and CDDP but not 5-FU. These results indicate that electrochemotherapy may be a promising treatment modality against CRC.

Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers.

BACKGROUND: When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver. AIMS: Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis. METHODS: Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and D-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coli beta-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of the lacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined. RESULTS: Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers. CONCLUSIONS: Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.

Particle-mediated gene transfer into murine livers using a newly developed gene gun.

Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

A novel approach for inducing enhanced and selective transgene expression in hepatocellular-carcinoma cells.

Utility of the alpha-fetoprotein (afp) promoter for gene therapy against hepatocellular carcinoma (HCC) is limited because of the weak promoter activity. To circumvent this, the 5.1-kb 5;-flanking sequence of the human afp gene including the entire enhancer and silencer regions as well as the promoter region was employed for achieving strong, HCC-selective transgene expression. To thoroughly inhibit the promoter activity of the 5;-flanking sequence of the human afp gene, the afp 5;-flanking region was inserted downstream of the human interleukin-2 (il-2) gene controlled by the simian-virus-40 (SV40) early promoter. il-2-production ability of HCC cells transduced with the construct was significantly enhanced compared with that transduced with the same construct lacking the afp 5;-flanking region. Importantly, il-2 production of non-HCC cells was substantially inhibited by the addition of the afp 5;-flanking region to the construct. When the afp 5;-flanking region was inserted downstream of the human tumor-necrosis-factor-alpha (tnf-alpha) gene controlled by the retrovirus long-terminal-repeat (LTR) enhancer/promoter, tnf-alpha production ability of HCC cells was significantly enhanced and that of non-HCC cells was significantly suppressed compared with that transduced with the same construct lacking the afp 5;-flanking region. Our results indicated that the afp 5;-flanking region gave the enhanced HCC-selective activity to the non-tissue specific SV40 early promoter and LTR enhancer/promoter. It is essential for successful gene therapy to induce strong, target-cell-selective transgene expression. This novel strategy, therefore, may contribute to the establishment of HCC-selective cancer gene therapy.

Electrochemotherapy can eradicate established colorectal carcinoma and leaves a systemic protective memory in mice.

Mice bearing subcutaneously established colorectal carcinoma (CRC) were given intratumoral, intravenous or intraperitoneal injection of various doses of bleomycin (BLM), followed by the delivery of direct current, square wave electric pulses to the tumor. Approximately 50% of animals treated with electrochemotherapy with BLM had completely eradicated established CRC tumors. Importantly, it was shown that CRC-specific cytotoxic T lymphocytes were elicited in the spleens of cured animals, resulting in the protection of the rechallenge with CRC. These results indicate that electrochemotherapy with BLM is promising for the treatment of metastatic CRC as well as the original lesion.

Immunomodulation with FK506 around the time of intravenous re-administration of an adenoviral vector facilitates gene transfer into primed rat liver.

Although adenoviruses are an attractive vehicle for gene transfer into tissues including various tumors, in vivo adenoviral administration elicits a neutralizing antibody response which eliminates or substantially reduces the efficacy of subsequent treatments. Transiently immunosuppressive strategies at the time of initial adenoviral exposure have shown to prevent the formation of neutralizing antibodies and permit the successful adenoviral readministration in animals. Initial treatment in humans may, however, correspond to adenoviral readministration into animals, because the exposure to wild-type adenoviruses is common in humans. In the present study, we infused Adex1CAlacZ adenoviruses carrying the lacZ gene into the tail vein of rats, and examined whether a transient treatment with the immunosuppressant FK506 around the time of i.v. readministration of adenoviruses could induce the re-expression of the lacZ gene in animals primed with adenoviruses. Although i.v. infusion of adenoviruses carrying the lacZ gene resulted in transiently high levels of transgene expression in rat liver, i.v. reinfusion of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with FK506 around the time of adenoviral reinfusion, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral reinfusion were significantly suppressed, and re-expression of the transgene was achievable.

Electrochemotherapy with bleomycin against colorectal carcinoma in a mouse model: evaluations of the dose and administration route of the drug and the electric field intensity.

Effectiveness of electrochemotherapy against colorectal carcinoma (CRC) was evaluated in a mouse model. When mice with a subcutaneously established CRC tumor were administered intratumorally, intravenously or intraperitoneally with bleomycin (BLM) ranging from 1/50 to 1/2 of the 50% lethal dose, significant suppression of tumor development and even some cures were observed. When various electric field intensities ranging from 500 to 2,000 V/cm were applied for electrochemotherapy with BLM, all treatment protocols were similarly effective. Furthermore, when electrochemotherapy with the lowest dose of BLM and the lowest electric field intensity was repeated, complete cures of CRC were achieved in all animals.

Comparison of gene therapy with the herpes simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma.

BACKGROUND: Bystander effects induced by suicide gene/prodrug systems play an essential role in achieving successful antitumor effects. Although it has been shown in several in vitro studies that the bacterial cytosine deaminase (CD) gene/5-fluorocytosine (5-FC) system is superior to the herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system, we examined here which suicide gene system was more promising in vivo for the treatment of hepatocellular carcinoma (HCC). METHODS: BNL1ME A.7R.1 murine HCC cells were retrovirally transduced with the HSV-TK or CD gene, and bystander effects caused by the appropriate prodrug treatment were examined not only in vitro but also in vivo. RESULTS: The CD/5-FC system was superior to the HSV-TK/GCV system in HCC cell elimination in vitro. The bystander effect of the HSV-TK/GCV was shown to be substantially dependent on cell-to-cell contact, whereas that of the CD/5-FC was not. However, antitumor effects on HCC and tumor immunity to parental HCC induced by the HSV-TK/GCV system were not inferior and even superior to those induced by the CD/5-FC system. Bystander effects induced by the suicide gene/prodrug systems in immunocompetent syngeneic mice were much more profound than those induced in vitro. However, significant bystander effects were not observed in athymic nude mice. CONCLUSIONS: These results suggest that both HSV-TK/GCV and CD/5-FC systems are useful for the treatment of HCC. The results also suggest that T-cell-mediated immune responses elicited by the suicide gene/prodrug systems play a substantial role in antitumor effects in vivo.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver.

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.