In vivo enhancement of herpes simplex virus thymidine kinase/ganciclovir cancer gene therapy with polyamine biosynthesis inhibition

We have earlier demonstrated that inhibition of polyamine biosynthesis with difluoromethylornithine (DFMO) can be used to enhance the cytotoxicity of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in different tumor cell lines. Here, the utility of this treatment combination was tested in vivo in a nude mouse tumor model. First, the effect of DFMO was verified by treating mice bearing subcutaneous 9L rat glioma tumors with 2% DFMO in drinking water. The drug treatment induced almost complete suppression of ornithine decarboxylase activity, and as a result, a strong decrease in intratumoral putrescine and spermidine concentrations, which were normalized 4 days after drug removal. Consequently, the tumors displayed a significant reduction in the proliferation activity that was increased to 20% higher than the normal level at day 4 and returned to normal level 7 days after DFMO removal. Next, 9L tumors with 30% of TK-GFP fusion gene positive cells were induced and the animals were given DFMO and GCV in 2 treatment schemes, with the drug administration periods overlapping either 5 or 2 days. The analysis of tumor size at the end of the treatment revealed that DFMO can enhance HSV-TK/GCV cytotoxicity when the overlap between DFMO and GCV was 5 days, but the result was not significant. However, the 2-day overlap scheme yielded a significantly (p < 0.05, ANOVA) enhanced antitumor effect. In conclusion, the data here confirms that a novel combination of 2 clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV suicide gene therapy, can be used synergistically in vivo.     

Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir

Transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSVtk-negative wild-type cells are killed by GCV, as are HSVtk-expressing cells. Our previous study demonstrated that intracranial 9L gliomas could be efficiently treated due to this bystander effect by injecting the 9L glioma cells transduced with the HSVtk gene in the vicinity of the preimplanted wild-type 9L glioma and then administering GCV. For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtkgene-transduced allogeneic C6 glioma cells (C6tk) instead of syngeneic 9L glioma cells transduced with the HSVtk gene. Fisher rats were implanted intracranially with wild-type 9L glioma cells, subsequently injected with C6tk cells at the same brain coordinate, and thereafter treated with GCV or saline. When the rats were treated with GCV, a significant retardation of tumor growth was observed by serial magnetic resonance imaging, although this growth retardation was less prominent than that observed with 9L glioma cells transduced with the HSVtk gene; consequently, survival was prolonged (P < .01). Tumors that received C6tk cells contained almost no HSVtk-positive cells after treatment with GCV. Rejection of allogeneic tumor cells, although possibly incomplete in the brain, can also contribute to the safety of this therapeutic strategy.     

Immunotherapy of murine prostate cancer using whole tumor cells killed ex vivo by herpes simplex viral thymidine kinase/ganciclovir suicide gene therapy

Whole cell cancer vaccines are currently under clinical evaluation. Their immunogenicity may depend on the mode of death of the vaccine cells prior to uptake by professional antigen-presenting cells and crosspriming of T cells. Destruction of tumor in vivo by genetic prodrug activation therapy leads to a marked local and systemic immune response, local T-cell infiltration and the establishment of T-cell memory. We postulated that this immunostimulation may be due to induction of danger signals and the inherent immunogenicity of products of HSVtk/ganciclovir kill. Using established models of murine prostate cancer, we have evaluated the efficacy of anti-tumor vaccines comprising irradiated allogeneic or autologous whole cells expressing HSVtK, which are first killed in vitro by prodrug activation using ganciclovir. HSVtk/ganciclovir-induced cell kill was through the induction of apoptosis. The vaccine was found to be effective in both models and superior to traditional irradiated whole tumor cells even after single doses. Protection against tumor challenge was associated with marked proliferative and Th1 cytokine responses. This approach would be applicable clinically in terms of ease of vaccine production, safety, storage and avoidance of potential toxicities of in vivo gene transfer.     

Herpes simplex virus thymidine kinase gene transduction enhances tumor growth rate and cyclooxygenase-2 expression in murine colon cancer cells

Transduction of tumor cells with herpes simplex virus thymidine kinase (HSV-tk) gene and subsequent treatment with the prodrug ganciclovir (GCV) is the most common system utilized to date for "suicide" gene therapy of cancer. In the current report, we show that HSV-tk gene transduction enhances tumor growth rate of murine colon cancer cells, that are implanted subcutaneously in syngeneic mice, and enhances cyclooxygenase-2 (COX-2) protein expression and prostaglandin E(2) (PGE(2)) release in vitro and in vivo. It is further shown that the observed phenomenon is related to the presence of the HSV-tk sequence insert in the retroviral vector used for HSV-tk gene delivery. Transduction of murine colon cancer cells with control vector, carrying the neomycin-resistance gene alone, failed to increase tumor growth rate and COX-2 protein expression or PGE(2) production. On the contrary, it even decreased tumor growth, COX-2 protein expression and PGE(2.) The growth rate of HSV-tk-transduced murine tumors was significantly reduced by treatment with the selective COX-2 inhibitor nimesulide. Additionally, we demonstrate herein that both enhanced growth rate of HSV-tk-transduced murine tumors and increased levels of PGE(2) in HSV-tk-transduced cells persist upon the development of GCV resistance. Taken together, these results provide a better understanding of the direct effect of HSV-tk gene transduction on tumor cell biology and target tumor development.     

Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.     

Temozolomide enhances herpes simplex virus thymidine kinase/ganciclovir therapy of malignant glioma

Gene therapy for malignant glioma with the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is already in the stage of clinical trials, but still needs major improvement to achieve greater clinical efficacy. The aim of this study was to determine whether combining HSV-tk/GCV gene therapy with temozolomide (TMZ), an alkylating drug clinically proven to be efficient in recurrent high-grade gliomas, would result in enhanced antitumor effect in malignant glioma in culture and in vivo. Human U87MG glioblastoma (GBM) cells with or without expression of HSV-tk were treated with different concentrations of GCV, TMZ, or both drugs. Cell viability was accessed by an automated microplate assay (MTT). The isobologram method and the combination index (CI) method of Chou-Talalay were used to measure the interactions between the two drugs when applied simultaneously. U87-tk and control U87 cells (5x10(6) each) were implanted in the flanks of nude mice, and animals were treated with GCV or TMZ or with both drugs. All tumors were measured and weighed at specified time points. IC(50) for GCV was 511 microM in control U87 cells and 14.3 microM in U87-tk cells, resulting in 35.7-fold increase of toxicity in the HSV-tk-expressing cells. TMZ had an IC(50) of 20.2 mM in control cells and 2.35 mM in U87-tk cells, resulting in 8.6-fold increase in sensitivity of the HSV-tk-expressing cells. TMZ and HSV-tk/GCV actions were synergistic (CI<1) in both control and U87-tk cells with higher synergism in U87-tk cells at high effect levels. Tumors expressing HSV-tk and treated with TMZ and GCV were significantly smaller than those treated by TMZ, but not by GCV. There was also a significant difference between the weight of HSV-tk expressing versus control tumors treated with TMZ, with GCV, or with both drugs. These data demonstrate synergism between HSV-tk/GCV and TMZ and higher sensitivity against TMZ in HSV-tk-expressing GBM cells. The potential importance for clinical studies combining both local tumor gene therapy and systemic chemotherapy should be explored further.     

Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir

Tumor targeting is an important issue in cancer gene therapy. We have developed a gene transfection method, based on light-inducible photochemical internalization (PCI) of a transgene, to improve gene delivery and expression selectively in illuminated areas, for example, in tumors. In the present work, we demonstrate that PCI improved the nonviral vector polyethylenimine (PEI)-mediated transfection of a therapeutic gene, the 'suicide' gene encoding herpes simplex virus thymidine kinase (HSVtk). In U87MG glioblastoma cells in vitro, the photochemical treatment stimulated expression of the HSVtk transgene, and, consequently, enhanced cell killing by the subsequent treatment with the prodrug ganciclovir (GCV). When relatively low doses of DNA (1 microg/ml) and the PEI vector (N/P 4) were used, HSVtk gene transfection followed by the GCV treatment did not have an effect on cell survival unless the photochemical treatment was performed, which potentiated the cytotoxicity to 90%. These findings indicate that photochemical transfection allows: (i) selective enhancement in gene expression and gene-mediated biological effects (cell killing by the Hsvtk/GCV approach) in response to illumination; (ii) the use of low, suboptimal for the nonviral transfection methods without PCI, doses of both DNA and the vector, which may be relevant and advantageous for therapeutic gene transfer in vivo.     

Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.     

Potentiation of ganciclovir toxicity in the herpes simplex virus thymidine kinase/ganciclovir administration system by ponicidin

The herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) administration system is commonly used in gene therapy trials. We have evaluated the effect of ponicidin, a diterpenoid isolated from a plant, Rabdosia ternifolia, on the cell-killing activity of the anti-herpes drugs acyclovir (ACV) and GCV. Ponicidin preferentially activated HSV-1-specific TK but not cellular kinases. In HSV-infected cells, ponicidin significantly accumulated the phosphorylated metabolites of GCV and suppressed the extracellular release of GCV. These data suggested that the cytotoxicities of ACV and GCV in HSV-TK-expressing cells might be potentiated by ponicidin. After transfected with the HSV-1 TK gene, COS-1 and several human cancer cells became highly sensitive to the cytotoxic properties of the nucleoside analogs. When ponicidin at the concentration without antiviral activities (0.2 microg/mL) was combined with ACV or GCV, the cytotoxic levels in HSV-TK-expressing cells were enhanced by 3- to 87-fold and 5- to 52-fold, respectively, compared with the nucleoside alone. When the stability of the bioactivity of ponicidin in the blood of mice was evaluated, the substance showed relatively long-lasting effects on the potentiation of the anti-herpetic and cytotoxic activities of GCV after intravenous administration. These data suggest that the combined use of ponicidin with GCV will be effective for cancer gene therapy, because high cytotoxicity in viral TK-expressing cells should yield more rapid and enhanced tumor elimination.     

Effects of retroviral-mediated herpes simplex virus thymidine kinase gene transfer to murine neuroblastoma cell lines in vitro and in vivo

Selective introduction of genes conferring chemosensitivity on proliferating tumor cells can be used to treat cancer. We investigated the efficacy of retrovirus-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell lines (neuro-2a) in vitro and in vivo. Retrovirus-mediated HSV-TK gene transfer to the neuro-2a cells resulted in sensitivity to ganciclovir (GCV) in vitro. In A/J mice, tumors produced from HSV-TK transduced neuro-2a cells regressed after GCV treatment. Intratumoral injection of recombinant retrovirus expressing HSV-TK gene also inhibited growth of the tumor established in A/J mice. These results demonstrate that HSV-TK gene therapy might be a feasible approach for inhibiting the growth of neuroblastoma.     

Rat glioma cell death induced by cationic liposome-mediated transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir treatment

BACKGROUND AND OBJECTIVES: We studied antitumor effects and cell death induced by cationic liposome-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir treatment in cultured rat T9 glioma cells and in experimental gliomas produced from this cell line. METHODS: To transfer genes we used small unilamellar cationic liposomes containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride. Video-enhanced contrast differential interference contrast microscopy was used for morphologic observations of cultured cells. RESULTS: When we treated the cells or implanted gliomas with the liposomes and ganciclovir, a strong effect was seen against tumor cells, and survival of tumor-implanted rats was increased. Morphologically, cell death observed after HSV-tk gene/liposome and ganciclovir treatment in the cultured glioma cells included both apoptosis and necrosis. CONCLUSIONS: Introduction of the HSV-tk gene in a DNA-liposome complex followed by ganciclovir treatment induced both apoptosis and necrosis, which together resulted in a potent antitumor effect.     

Sites of integration of herpes simplex virus type-2 thymidine kinase gene in human chromosomes.

Isozyme analyses have been carried out to investigate the sites of integration of the herpes simplex type 2 (HSV-2) thymidine kinase (TK) gene in biochemically transformed human [HeLa(BU25)/HSV-2–6 Cl 4] cells. Extracts were prepared from He La (BU25)/HSV-2–6 Cl 4 cells and from human—mouse somatic cell hybrids, which were obtained by fusing the biochemically transformed human cells with TK-deficient mouse [LM(TK^?)] cells, and were assayed for 26 isozymes representing markers for 20 human chromosomes. The isozyme analyses were generally consistent with previous karyotype studies, which revealed that the HSV-2 TK gene was associated with an isochromosome, designated M13, formed from the short arm of human X chromosome in human—mouse hybrid lines HL/2 to HL/7, but with human chromosome 17 containing a trans-location on the short arm in hybrid line HL/1 and its TK-positive subclones. The isozyme analyses also indicated that the translocation on the short arm of human chromosome 17 in hybrid line HL/1 was probably derived from human chromosome 21. Hybrid line HL/1 and its TK-positive subclones expressed a human—mouse heteropolymeric form of superoxide dismutase, a marker for human chromosome 21, but bromodeoxyuridine-resistant, TK-negative subclones of HL/1, and hybrid lines HL/3 to HL/6, which did not contain the modified chromosome 17, failed to express the human—mouse heteropolymeric form of superoxide dismutase. The human galactokinase isozyme, which is coded by a gene mapping close to the cytosol TK gene on human chromosome 17, was detected in extracts of TK-positive HeLa S3, TK-deficient HeLa(BU25), and biochemically transformed HeLa(BU25)/HSV-2–6 Cl 4 cells, but not in extracts prepared from human—mouse hybrid line HL/1 and its subclones. These observations suggest that the gene for human galactokinase was either deleted or inactivated in HL/1 and its subclones, perhaps as a result of the translocation to chromosome 17.     

Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkappaB and 80-85% for TOZ.1. TOIkappaB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.     

Association of herpes simplex thymidine kinase gene with chromosome No. 18 in transformed human cells

The herpes simplex virus type-2 (HSV-2)-transformed human cell line HB-2-3 was fused with thymidine kinase (TK)-deficient mouse cells [LM(TK-)], and 12 independent hybrids were isolated with the use of the HAT (hypoxanthine, amethopterin, and thymidine)-ouabain selection system. Discontinuous polyacrylamide gel electrophoresis studies demonstrated that the HSV-2-specific TK was the selected enzyme in the hybrids. Isoenzyme analysis and karyotyping were used in the analysis of the hybrids for the presence of human chromosomes. All 12 hybrids contained human chromosome No. 18 and the enzyme peptidase A, which is encoded by a gene on this chromosome. Hybrids were exposed to bromodeoxyuridine (BrdUrd) as a means of selection for cells that had lost HSV-2 TK activity. Isoenzyme and karyotyping data obtained from 33 BrdUrd-resistant sublines were consistent with the hypothesis that the HSV-2 TK gene is associated with chromosome No. 18 in the HB-2-3 cell line.     

Antitumor effects on human melanoma xenografts of an amplicon vector transducing the herpes thymidine kinase gene followed by ganciclovir

Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potentially useful gene delivery vector for gene therapy due to its high efficiency of in vivo transduction. The helper virus-dependent, HSV- 1 amplicon vectors were developed for easier operation and their larger capacity. In this study, the herpes simplex virus type-1 thymidine kinase (HSVtk) gene was cloned into the pHE700 amplicon vector to make an HE7tk vector and used for in vivo gene delivery. Human melanoma xenografts were established in athymic nude mice. Tumors were injected directly with HE7tk vector alone, HE7tk vector followed by ganciclovir (GCV), or a pHE700 amplicon vector carrying a green fluorescent protein (HE7GFP) gene followed by GCV. Efficient HSVtk transgene expression was found in the tumor 3 days after injection. Animals transduced with HE7tk followed by GCV had minimal tumor growth (P < .01 ). Animals that received either HE7tk vector without GCV or HE7GFP vector with GCV had some reduction in tumor growth compared to animals that were injected with buffer only. These data indicate that replication-defective HSV-1 amplicon vectors can be used effectively to deliver transgenes into solid tumors in vivo.     

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy in BT4C rat glioma model

Adenovirus (Adv)-mediated herpes simplex virus thymidine kinase (adv/tk) gene therapy combined with ganciclovir (GCV) medication is a promising approach for the treatment of malignant glioma. However, optimal administration and the effect of possible adjuvant treatments have not been fully examined. In the present study, we examined the efficacy of adv/tk/GCV gene therapy in a syngeneic BT4C rat malignant glioma model, either as a single administration or given as three injections during three consecutive days. The effect of combined adv-mediated macrophage colony-stimulating factor (MCSF) and adv/tk gene transfer was also studied. BT4C malignant glioma cells were injected into the right corpus callosum of BDIX rats (n=112). Before gene therapy, the presence of tumors was verified by MRI. The rats were divided into eight groups as follows: group I (n=20) received a single adv/tk gene transfer (total dose 4x10(8) pfu) and GCV treatment for 14 days; group II (n=5) received the same gene transfer without GCV; group III (n=28) received three adv/tk injections (total dose 4x10(8) pfu) on three consecutive days and GCV for 14 days; group IV (n=5) received three similar adv/tk injections without GCV medication; group V (n=13) received three adv/MCSF injections (total dose 2x10(8) pfu) on three consecutive days and GCV medication; group VI (n=12) received three adv/tk and adv/MCSF (total dose 6x10(8) pfu) injections on three consecutive days followed by GCV medication; and group VII (n=12) the same treatment without GCV. Group VIII (n=17) consisted of wild-type BT4C malignant glioma tumors without any treatment. Treatment effect and tissue responses were characterized by general histology, immunohistochemistry, MRI, and survival of the study groups. The best treatment effect and survival was found in rats treated with adv/tk gene transfer once a day for three consecutive days (P<.05). No improvement of the treatment effect was seen after the combined adv/tk and adv/MCSF gene transfer compared with the repeated adv/tk gene transfer. The results show that 20% of the rats can be cured (survival >6 months) after optimized adv/tk gene therapy. It is concluded that repeated intratumoral administration of adv/tk is a promising approach for the treatment of malignant glioma tumors in vivo.     

缺氧对血管内皮生长因子启动子-胸苷激酶系统杀伤肝癌细胞系的增强作用

目的：探讨缺氧对腺病毒介导的血管内皮生长因子启动子－胸苷激酶系统（AdVEGF-tk)对肝癌细胞系HepG2选择性杀伤活性的增强作用。方法：采用AdEasy system构建重组腺病毒载体AdVEGF-tk和AdVEGF-GFP,在293细胞中包装,扩增后,分别感染L02（正常肝细胞系）和HepG2,A VEGF-GFP感染细胞经正常培养24h后,在荧光显微镜下观察其荧光蛋白表达情况;AdVEGF-tk感染细胞在缺氧或不缺氧条件下培养,并给予不同浓度的丙氧鸟苷（GCV）处理,用MTT法检测感染细胞的增殖情况,结果：AdVEGF-GFP感染的L02细胞仅见稀散的荧光,而AdVEGF-GFP感染的HepG2细胞则出现大量荧光,感染AdVEGF-tk后,当感染指数（MOI）为100目GCV浓度为10ug/ml时,L02细胞在不缺氧条件下对GCV几乎不敏感,但在缺氧条件下,70％以上的细胞被杀死,而HepG2细胞即便在不缺氧培养条件下也有60％以上的细胞被杀死,缺氧则使80％以上细胞被杀死,结论：缺氧可加强腺病毒介导的AdVEGF-tk系统对体外培养肝癌细胞的选择性杀伤作用。     

Cooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer

Adenovirus-mediated transduction of the herpes simplex thymidine kinase gene (HSV-tk) in conjunction with ganciclovir (GCV) has been shown to result in significant growth suppression and to enhance survival in a model of mouse prostate cancer. However, this therapeutic activity is not sustained, because in most cases tumors eventually regrow and ultimately cause the death of the host. Androgen ablation, an inducer of apoptosis in prostate cells which is used widely as palliative therapy in patients with prostate cancer, was combined with HSV-tk plus GCV using an androgen-sensitive mouse prostate cancer cell line. The combination of castration and HSV-tk plus GCV led to markedly enhanced tumor growth suppression in both subcutaneous and orthotopic models compared with either treatment alone and resulted in an enhanced survival in which combination-treated animals lived twice as long as controls in the subcutaneous model and over 50% longer than controls in the orthotopic model. Further analysis of apoptotic activity demonstrated high levels of apoptosis only in combined androgen ablation and HSV-tk plus GCV-treated tumors after 14 days of growth in an androgen-depleted environment and 8 days after HSV-tk plus GCV therapy. At this time, the apoptotic index, but not the percent of necrotic tissue, was significantly higher for combination therapy-treated tumors relative to control-treated tumors or either treatment alone. These data indicate that the therapeutic effects of androgen ablation and HSV-tk plus GCV are cooperative and that increased apoptosis may, in part, underlie these activities.