Human herpesvirus 8-encoded thymidine kinase and phosphotransferase homologues confer sensitivity to ganciclovir.
(17/2453)
Human herpesvirus 8 (HHV-8) sensitivity to the nucleoside analog ganciclovir (GCV) suggests the presence of a virally encoded kinase that catalyzes the initial phosphorylation of GCV. Analysis of the HHV-8 genome identified two candidate kinases: proteins encoded by open reading frame (ORF) 21, with homology to the herpesvirus thymidine kinases (TK), and ORF 36, with homology to the herpesvirus phosphotransferases (PT). Experiments presented here show that both ORF 21 and ORF 36 encode GCV kinase activities as demonstrated by GCV phosphorylation and GCV-mediated cell death. In both regards the PT homologue ORF 36 was more active than the TK homologue ORF 21. ORF 21, but not ORF 36, weakly sensitized cells to killing by penciclovir. Neither ORF sensitized cells to killing by (E)-5-(2-bromovinyl)-2'-deoxyuridine. (+info)
Repeated cycles of retrovirus-mediated HSVtk gene transfer plus ganciclovir increase survival of rats with peritoneal carcinomatosis.
(18/2453)
Peritoneal carcinomatosis is a common clinical situation that requires novel therapeutic approaches. We investigated the efficiency of an HSVtk gene therapy for the treatment of peritoneal carcinomatosis induced in syngeneic rats by DHD/K12 colon carcinoma cells. In this setting, the efficiency of two different retrovirus producing cell lines (GP+AmEnv12 and FLYA13) was compared. Rats treated with a single injection of retrovirus producing cells followed by a 5-day course of ganciclovir treatment showed an increased survival as compared with control animals. Animals treated with three injections of producing cells, each followed by a 4-5-day course of ganciclovir treatment, showed an increased survival as compared with control rats and with those treated with a single cycle of retrovirus producing cells plus ganciclovir. However, only a few animals remained tumor-free after day 180. There was no difference between the two producing cell lines in any of the experiments. RT-PCR demonstrated a faint expression of the tk transgene in the liver, spleen, epiploon, bowels and the lung of the animals injected with the HSVtk producing cells, reflecting most likely the transduction of only a limited number of cells. (+info)
Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma.
(19/2453)
We have previously demonstrated that transfected hepatocellular carcinoma cells (Hepa1-6) with one copy (pAGO) and two copies (pYED) of the HSVtk gene, using liposomes, induced cell death of untransfected cells in the presence of ganciclovir (GCV). This phenomenon is called the 'bystander effect'. To determine whether an elevated level of connexin43 increases the bystander effect, we have cotransfected Hepa1-6 cells with a plasmid containing the HSVtk gene driven by the alpha-fetoprotein promoter (pFTK) or pAGO or pYED and connexin43. The results showed that, after GCV treatment, the percentage of growth inhibition was higher (25-30%) in cells cotransfected with HSVtk and connexin43 than in cells transfected only with HSVtk gene. The IC50 of GCV on cells transfected with pFTK/Connexin43 was 17.85-fold lower than cells transfected with pFTK alone. To improve these results, stable connexin43 transduced Hepa1-6 cells were transfected with pFTK followed by GCV treatment. In this case, the cell growth was markedly inhibited as compared with parental cells. Furthermore, we have studied the correlation between the expression of the HSVtk and the connexin43 proteins. Using flow cytometric analysis, scrape loading/dye transfer and immunoblotting assay we found that the cells transfected separately by pAGO, pYED, pFTK and pLTR-Cx43 showed an increase of connexin43 protein. This study indicates that transfecting Hepa1-6 cells with both connexin43 and HSVtk genes up-regulates connexin43 expression which enhances the bystander effect and subsequently tumor cell death. (+info)
Intracerebral injection of adenovirus harboring the HSVtk gene combined with ganciclovir administration: toxicity study in nonhuman primates.
(20/2453)
A high dose (1-2.5 x 10(10) infectious units) of recombinant adenovirus harboring the herpes simplex thymidine kinase gene (IG.Ad.MLPI.TK) was injected into the white matter of the right frontal lobe in two rhesus monkeys (M. mulatta). Injection of the vector was followed by systemic ganciclovir administration (10 mg/kg per day) for 14 days. During treatment no clinical symptoms were observed. Histopathological analysis of the brain at day 18 showed a 5 mm necrotic area at the site of the virus injection. This area was invaded and surrounded by inflammatory cells and activated astrocytes (gliosis). Immunohistochemical analysis of the infiltrates revealed the presence of predominantly mononuclear cells. In the vicinity of the lesion perivascular cuffs were seen containing T lymphocytes and clusters of B lymphocytes. From this preclinical study we conclude that the toxicity of adenotk/GCV is acceptable and treatment of patients with malignant gliomas using this kind of therapy is feasible. However, careful dose finding in clinical studies is recommended. (+info)
Production and culture of HSVtk transduced suicidal lymphocytes induces variable changes in the lymphocyte subset composition.
(21/2453)
Allogeneic bone marrow transplant followed by donor lymphocyte infusion (DLI) is limited by T cell-mediated graft-versus-host disease (GVHD). A potential solution to alleviate uncontrolled GVHD is to create a controllable suicidal lymphocyte using retroviral transduction of the herpes simplex virus thymidine kinase gene (HSVtk) into the T cell graft. Should GVHD arise, the administration of ganciclovir (GCV) should eliminate the causal T cells. The culture conditions and expansion protocols required to produce suicidal lymphocytes may affect the composition of the T cell product. In this report we describe how T cells from individual donor samples respond to the same culture condition in highly varied ways. Among five donors, two demonstrated predominant expansion of CD4+ cells (with a decrease of CD8+), two donors resulted in predominately CD8+ cells, and one donor developed mainly dual positive CD8+/CD56+ cells. We observed a 20-fold expansion of T cells during the 14 day protocol. The function of the T cells was not affected by the transduction procedures (as tested by 51Cr release assays). In contrast to suicidal lymphocytes prepared using entire T cell populations, T cells pre-selected into CD3+/CD4+ or CD3+/CD8+ subpopulations prior to culture maintained their initial phenotype during the 14 day culture period, with little or no drift. Results from clinical trials using suicidal lymphocytes may be confounded by variance in lymphocyte subset compositions (LSC) and optimal use of suicidal lymphocytes may require separate culture and transduction to control the LSC delivered to the patient. (+info)
Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22.
(22/2453)
We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols. (+info)
Enhanced plasma cholesterol lowering effect of retrovirus-mediated LDL receptor gene transfer to WHHL rabbit liver after improved surgical technique and stimulation of hepatocyte proliferation by combined partial liver resection and thymidine kinase--ganciclovir treatment.
(23/2453)
In this study we report an improved method for in vivo gene transfer to liver. Repeated injections of Moloney murine leukemia virus-derived retroviruses containing LDL receptor cDNA were given to the portal vein in combination with a 10% partial liver resection and stimulation of hepatocyte proliferation by plasmid/liposome-mediated thymidine kinase gene transfer and ganciclovir treatment. The method was used for the treatment of LDL receptor deficiency in Watanabe heritable hyperlipidemic rabbits. We demonstrate an increase in hepatocyte proliferation index by thymidine kinase and ganciclovir treatment from 0.9 to 1.35% and a maximum of 35% decrease in total plasma cholesterol level 2-3 months after the gene transfer. A 20% decline was still present after a 52-week follow-up period. A 50% decrease was also observed in plasma triglycerides. Liver function tests indicated a transient increase in plasma alkaline phosphatase level up to 12 weeks after the gene transfer. In situ PCR and RT-PCR analyses indicated that the transgene was present in periportal areas and was transcribed to mRNA 1 week after the gene transfer. Because of the relatively simple and controllable technique we suggest that repeated retrovirus injections via a portal vein catheter together with the limited partial liver resection and plasmid/liposome-mediated thymidine kinase gene transfer-ganciclovir treatment may be used to improve the results of retrovirus-mediated liver gene therapy. (+info)
Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer.
(24/2453)
A major obstacle to the success of gene therapy strategies that directly target cancer cells is the poor vector distribution within solid tumors. To address this problem, we developed an E1b 55 kDa attenuated, replication-competent adenovirus (Ad.TKRC) which expresses the herpes simplex-1 thymidine kinase (HSVtk) gene to sensitize tumors to ganciclovir (GCV). Efficacy of this combined strategy was tested in nude mice with subcutaneous human A375 melanoma and ME180 cervical carcinomas. Intratumoral injection of a replication-defective adenoviral vector expressing HSVtk (Ad.TK) followed by GCV treatment resulted in doubling of the survival time of mice bearing A375 tumors and 20% long-term survival of mice with ME180 tumors. Treatment of tumors with Ad.TKRC without GCV resulted in a similar antitumor effect, confirming that the replicating vector has an oncolytic effect. When GCV was initiated 3 days after Ad.TKRC injection, survival of mice with each tumor type was greatly prolonged, with 60% of animals with ME180 tumors surviving for over 160 days. These results confirm that both the oncolysis caused by a replicating virus and suicide/prodrug gene therapy with HSVtk/GCV have potent antitumor effects. When combined, these two approaches are complementary resulting in a significantly improved treatment outcome. (+info)