Animal experiment on gene therapy of ovarian cancer by adenovirus-mediated thymidine kinase gene transduction and ganciclovir administration in vivo

OBJECTIVE: The efficacy and toxicity of adenovirus-mediated transduction of herpes simplex virus thymidine kinase gene started by Rous sarcoma virus (ADV/RSV-tk) followed by administration of ganciclovir (GCV) were studied in vivo. METHODS: An animal model of human epithelial ovarian cancer was established in nude mice using the serous ovarian adenocarcinoma cell lines Ov-ca-2774, then mice were treated by ADV/RSV-Tk and GCV, or GCV and HSV-tk respectively. The average survival time of mice and toxicity were assessed. RESULTS: The mice treated with GCV or HSV tk alone died from 14.4 +/- 1.7 to 19.3 +/- 3.5 days after treatment. The survival time had no difference with control group. The mice treated with ADV/RSV-tk followed by GCV lived at least two times longer than controls and the difference in both groups was significant. The earlier the treatment began, the longer the average survival time was. Treatment efficacy was dependent on dose of ADV/RSV-tk and tumor burden of mice. CONCLUSION: ADV/RSV-tk gene therapy is a safe and efficient approach to ovarian cancer treatment in the experiment.     

The treatment of malignant mesothelioma with a gene modified cancer cell line: a phase I study

Malignant mesothelioma is a tumor of the pleura for which there is no satisfactory treatment. It is almost universally fatal, regardless of the stage of the tumor at the time of diagnosis. Current treatment modalities include surgery, chemotherapy, and radiation therapy, although in some series none of these modalities is superior to no treatment at all. Because of the dismal prognosis for patients with malignant mesothelioma, a new mode of treatment is desperately needed. A promising area of research into the treatment of various malignancies is gene therapy. Recent studies have demonstrated the utility of exposing tumor cells to cells transduced to express the Herpes simplex virus gene for thymidine kinase (HSV-tk). By virtue of their expression of HSV-tk, the transduced cells are rendered susceptible to the antiviral drug, ganciclovir (GCV). and nearby tumor cells are killed by a phenomenon termed the bystander effect. In this protocol we propose a Phase I trial to study the safety and determine the maximal tolerated dose of an HSV-tk-transduced ovarian cancer cell line (PA1-STK cells) infused into the pleural cavities of patients with malignant pleural mesothelioma, followed by systemic administration of ganciclovir. The hope is that administration of ganciclovir will result in killing of the HSV-tk transduced ovarian cancer cells as well as the nearby malignant mesothelioma cells. This is a standard dose-escalation protocol.     

Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer

Metastases of breast cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of suicide gene therapy in metastatic breast cancer, we used the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) administration to treat breast cancer, generated by an adenocarcinoma cell line MOD in syngeneic mice. The bystander effect of HSV-tk + GCV on tumor cell killing was illustrated by demonstrating complete regression of subcutaneous tumors consisting of 90% parental tumor cells and 10% HSV-tk transformed tumor cells. To establish a model of breast cancer metastases in the liver, tumors were generated by intra-hepatic implantation of MOD cells in syngeneic animals. Two weeks after tumor cell implantation, replication defective adenoviral vectors expressing HSV-tk (ADV.tk), or beta-galactosidease (ADV. beta-Gal) were injected intratumorally, followed by buffer or GCV administration. Treatment with ADV.tk + GCV resulted in significant regression of tumor (P < .001), as assessed by computerized morphometric analysis of residual tumor. This was reflected as a significant prolongation of survival in treated animals (P < .001). These results demonstrate that ADV-mediated suicide gene therapy in vivo can be incorporated in a comprehensive treatment strategy for liver metastases of breast cancer.     

Enhancement of tumor killing using a combination of tumor immunization and HSV-tk suicide gene therapy

Tumor cells genetically modified with the herpes simplex virus thymidine kinase (HSV-tk) gene in combination with ganciclovir (GCV) demonstrate a "bystander effect". Previous attempts to enhance the bystander tumor killing by combining cytokine genes with HSV-tk/GCV have met with varying results. The present study was designed to determine the effects of tumor immunization in combination with HSV-tk gene-modified tumor cells and GCV on tumor killing and to determine if the bystander tumor killing could be enhanced. Tumor-bearing mice immunized with syngeneic tumor (KBALB) prior to treatment with an i.p. injection of xenogeneic HSV-tk gene-modified tumor cells (PA-1STK) had prolonged animal survival (group 4, 56.4 days). In contrast, unimmunized tumor-bearing mice (group 2) or tumor-bearing mice immunized to the xenogeneic PA-1STK tumor cells (group 5) showed a mean survival of about 27 days after receiving an i.p. injection of PA-1STK cells and GCV. Control groups, which were either not immunized and did not receive HSV-tk cells (group 1) or immunized but treated only with GCV (group 3) showed short survival (16-18 days). Analysis of tumors for cytokine mRNA expression revealed increased TNF-alpha and IL-1alpha mRNA expression in group 4 mice. Furthermore, IL-2 mRNA expression was detectable on days 2 and 4 only in group 4 mice. Immunophenotypic analysis for tumor-infiltrating lymphocytes demonstrated an increase in macrophage (4%, p = 0.0001) and T cells (1.8%, p < 0.001) in group 4 mice with an enhanced T-cell response as compared with mice from groups 1, 2 and 3. Our results demonstrate that tumor immunization combined with HSV-tk/GCV treatment results in increased animal survival with enhanced immune response. Furthermore, the cytokine milieu observed in the present study can modulate the tumor micro-environment in vivo from one that is immunosuppressive to one that is immune-stimulatory.     

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.     

Limited efficacy of the HSV-TK/GCV system for gene therapy of malignant gliomas and perspectives for the combined transduction of the interleukin-4 gene

The growth of U-87 or C6 gliomas co-implanted in nude mice with retroviral producer cells (VPC) expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene is only partially impaired by treatment with ganciclovir (GCV). The effect of GCV is even less evident when C6 and VPC are co-implanted into the rat brain. Furthermore, tumors from C6 cells carrying the HSV-tk gene are not eradicated by GCV, although they remain sensitive to GCV when replated in vitro. These limits of the HSV-tk/GCV system in glioma gene therapy may be due to insufficient gene transfer and/or insufficient delivery of GCV to glioma cells. Combination of HSV-tk and one or more cytokines may improve the antitumor efficacy. Among cytokines, interleukin-4 (IL-4) has already been shown to be active against gliomas. In nude mice, GCV treatment inhibited tumor growth more effectively after co-injection of C6 cells with a mixture of VPC transducing IL-4 and HSV-tk genes than after co-injection with either IL-4 or HSV-tk VPC only. In immunocompetent Sprague-Dawley rats, co-injection of IL-4 VPC and C6 cells was also effective in inhibiting the growth of C6 brain tumors, 38% of the animals surviving for at least 2 months. Furthermore, increased and prolonged antitumor efficacy was obtained by transducing both IL-4 and HSV-tk genes.     

Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV +/- apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60-70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells.     

Recent advances in dementia research in Japan: non-Alzheimer-type degenerative dementias

Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r = 0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk.     

Inhibitory effect of ganciclovir on the HSV1-tk positive subcutaneous tumors transplanted with human ovarian cancer in nude mice

OBJECTIVE: To investigate the inhibitory effect in vivo of ganciclovir (GCV) on the growth of human ovarian cancer cells (AO) transducted with the thymidine kinase gene of herpes simplex virus I type (HSV1-tk). METHODS: Tumors were induced in nude mice by subcutaneous injection of AO cells and AO cells carried with HSV1-tk gene from China strain (AO/HSV1-tk cells). When the growing tumors were visible, GCV was injected daily into the peritoneum of the nude mice. RESULTS: The average weights of survived AO/HSV1-tkc tumors and AO tumors treated with GCV were 0.087 +/- 0.036 g and 0.661 +/- 0.260 g respectively. Most of the survived AO/HSV1-tkc cells treated with GCV were characterized by hypertrophy and necrosis, but their nuclear chromatins predominantely took the forms of heterchromatins. CONCLUSIONS: GCV could effectively inhibit the growth of HSV1-tk positive human ovarian cancer cells in vivo, but the nuclei of the survival tumor cells appeared to proliferate actively. As the same results of in vitro experiments, this may suggest that HSV1-tk/GCV gene therapeutic system might be combined with S-phase chemotherapy to increase the long-term effect.     

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.     

Isolation of human mannan binding lectin, serum amyloid P component and related factors from Cohn fraction III

BACKGROUND: The suicide gene and prodrug, herpes simplex thymidine kinase (HStk) and ganciclovir (GCV), are now in clinical trials for recurrent malignancies. METHODS: We evaluated in vitro and in vivo efficacy of HStk gene transfer and GCV treatment of colonic adenocarcinoma in a syngeneic murine model. RESULTS: In vitro analysis demonstrated that CT-26 adenocarcinoma cells transduced with LTKOSN.2 retroviral vector inhibited the proliferation of wild-type CT-26 (nontransduced) cells after GCV exposure. Cooperative killing with HStk gene therapy was shown in vivo, mixtures of HStk CT-26 transduced cells (CT-26 TK), and nontransduced (CT-26 NV) cells and tumors containing only 9% CT-26 TK cells demonstrated complete regression after GCV (100 mg/kg). CONCLUSIONS: This in vitro and in vivo demonstration suggests that metabolic cooperation permits destruction of tumors even when gene transfer is effective only to a relatively small portion of the tumor. These important results suggest new avenues can be developed for the treatment of this lethal malignancy.     

Combination therapy with suicide and cytokine genes for hepatic metastases of lung cancer

Metastases of lung cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of gene therapy in metastatic lung cancer, we used adenoviral (ADV) mediated transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene and the cytokine gene interleukin-2 (IL-2) to treat a murine model of metastatic lung cancer in the liver. Hepatic metastases were established by intrahepatic implantation of LL2 cells in syngeneic recipient mice. One week after tumor implantation, various replication defective ADV vectors were injected intratumorally. Treatment with a vector expressing the HSV-tk followed by ganciclovir administration with ADV.tk resulted in significant regression of tumor (p<0.01) as well as prolongation of survival (p<0.001). While a vector expressing mouse IL-2 ADV.IL-2 alone was ineffective, combination therapy with HSV-tk resulted in further tumor regression and improvement of animal survival (p<0.05). These results demonstrate that suicide and cytokine genes can be utilized in combination to treat metastatic lung cancer in vivo.     

Mechanisms for ganciclovir resistance in gastrointestinal tumor cells transduced with a retroviral vector containing the herpes simplex virus thymidine kinase gene

Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tumor cells confers sensitivity to the cells to the viral drug ganciclovir (GCV). Although the efficacy of the HSV-TK/GCV approach is well studied, the mechanisms for the resistance of HSV-TK-transduced tumor cells to GCV are poorly understood. Here, we examined the mechanisms for GCV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines. Our results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines. GCV-resistant colonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-resistant colonies demonstrated that the HSV-TK gene was either partially or completely deleted from the resistant HSV-TK-transduced cells. In the HT-29 RM and MIAPACA-2 RM cells, a 220-bp deletion of the gene was found, whereas in the HT-29 R1-R5-resistant cells, the whole TK gene was found to be absent. Immunocytochemical studies using a polyclonal antibody to the TK protein demonstrated that the HSV-TK protein was absent in the GCV-resistant, HSV-TK-transduced cells. Transfection of the resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV. The presence of GCV-resistant cells was only demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect. Our results suggest that GCV resistance found in tumor cells transduced with a retroviral HSV-TK gene is due to the lack of a functional TK protein in the tumor cells rather than any intrinsic resistance of the cells to GCV. In tumor cells with a good bystander effect, the small percentage of TK-transduced cells that do not express the TK protein are probably killed by the bystander effect because GCV-resistant tumor cells were not found in these cell lines. GCV-resistant tumor cells were found only in tumor cell lines with a poor bystander effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.     

Variable efficiency of the thymidine kinase/ganciclovir system in human glioblastoma cell lines: implications for gene therapy

The gene therapy strategy using the hsvl-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells, is needed. After proposing sensitivity criteria for the TK/GCV system and for the bystander effect, based on the levels of GCV that can be reached in vivo, we studied seven human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. Among six human glioblastoma cell lines stably transfected with the TK gene, five were sensitive to TK/GCV, and two had a good in vitro bystander effect. The in vitro transfectability of the cell lines tested was low (< or = 1%) compared to that of an established animal cell line, C6 rat glioma, in which 20-30% of the cells can be transfected routinely. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is an urgent need for agents to increase transfection efficiency.     

Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.     

Shelf life of sliced raw potatoes of various cultivar varieties--substitution of bisulfites

Treatment of colorectal liver metastases with the HSVtk/GCV approach and adenoviral vectors is highly toxic. We present a nontoxic alternative using the cell type-specific CEA promoter instead of the widely used hCMV immediate-early promoter to drive tk gene expression in the context of a recombinant adenovirus. Analysis of CEA promoter-dependent tk gene expression showed significant activity of this promoter in several human and rat tumor-derived cell lines but not in rat primary hepatocytes and in mouse liver, whereas the CMV promoter was highly active in all cell types and tissues investigated. CEA promoter-dependent tk gene expression was sufficient to kill 100% of cancer cells in vitro, even if less than 10% were infected by the adenoviral vector, indicating a significant bystander effect. Moreover, treatment of subcutaneous tumors in SCID mice with Ad.CEA-tk led to a several-fold reduction of tumor growth, and tail vein injection of a high dose of Ad.CEA-tk caused no side-effects in the liver. The CMV promoter was more potent than the CEA promoter in mediating GCV sensitivity to cancer cells in vitro and in vivo, but even a 20-fold reduction of the dose of Ad.CMV-tk did not prevent its liver cell toxicity after systemic application to mice and still resulted in the death of all animals within 4 days after the start of GCV treatment. These results indicate that restriction of tk gene expression to tumor cells in the liver prevents systemic toxicity. Moreover, the CEA promoter is a safe and efficient tool for tumor cell-specific expression of suicide genes in the liver.     

Multicomponent gene therapy vaccines for lung cancer: effective eradication of established murine tumors in vivo with interleukin-7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo activated dendritic cells

Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.     

Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.     

Prospect of gene therapy for metastatic foci of gastric cancer

Surgical excision of both primary and metastatic tumors is the recommended therapy for gastric cancer. There are many cases of multiple metastasis to lymph nodes, disseminated metastasis of peritoneum, and post-operative systemic metastasis for which no surgical treatment is available. Cancer gene therapy is one of the promising new techniques. We injected the suicide gene, herpes simplex virus thymidine kinase (HSV-tk), directly into the tumor using gastrofiberscopy. Following GCV treatment caused tissue degeneration at the injection site and lymph nodes. These findings show that suicide gene therapy for gastric cancer may be effective for a metastatic tumor along with the primary tumor.     

Transcriptional activation of NF-kappa B activity by Epstein-Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases

Epstein-Barr virus (EBV) has been known to be associated with many malignant tumors, including nasopharyngeal carcinoma (NPC). Previous studies have indicated that an EBV-encoded oncoprotein, latent membrane protein 1 (LMP1), is expressed in many NPC tissues. LMP1 has been shown to stimulate HIV LTR through the two NF-kappa B binding sites within this promoter. In this study, we examined the feasibility of using this property of LMP1 as a therapeutic strategy for the treatment of NPC. This therapy consists of the preferential killing of the LMP1-expressing cells by gene transfer using the NF-kappa B-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. The 800-bp HIV-LTR, which contains two NF-kappa B binding sites, was used to drive the HSVtk gene. Stable C33A cell clones expressing the LMP1 and the HSVtk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP1 and the HSVtk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became palpable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily. Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP1 and the HSVtk genes, but not in mice carrying cells containing LMP1 or HSVtk alone. Our data indicate that the HSVtk gene expressed from a NF-kappa B-binding motif-containing promoter that is regulated by LMP1 may be used as an in vivo gene therapy strategy of EBV LMP1-expressing cancers such as NPC.