Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells.

Identification of polyamides that enhance adenovirus-mediated gene expression in the urothelium.

Adenovirus-mediated gene therapy of bladder diseases has been limited by the inability to transduce the urothelium successfully using adenoviral vectors. We have sought to identify agents that would increase adenovirus-mediated transgene expression in the bladder. We have utilized a rat model to screen compounds for their ability to enhance viral transgene expression in the rat bladder. Rats received intravesical administration of replication-deficient adenovirus (rAd) formulated in various agents, and transgene expression was evaluated after 48 h by determining the amount of lacZ expression in the luminal epithelium of the bladder. We report the identification of two different polyamides, each capable of dramatically increasing viral transgene expression in the bladder without causing detectable alteration of the umbrella cell layer of the urothelium. We have utilized a carcinogen-induced rat bladder tumor model to demonstrate that these polyamides are also capable of enhancing viral transgene expression in tumor tissue. The identification of these polyamides potentiates the use of adenovirus-mediated gene therapy for the treatment of superficial bladder cancer or other bladder diseases.

Ethanol improves adenovirus-mediated gene transfer and expression to the bladder epithelium of rodents.

OBJECTIVES: Replication deficient adenoviral vectors (rAds) are used as gene delivery systems that can efficiently transduce a variety of tissues and may be appropriate vectors to develop gene therapeutics for many urologic applications. However, the bladder epithelium has been shown to be highly resistant to transgene expression after intracystic administration. A potential explanation for this low gene transfer efficiency may be the protective structure of the urothelium. Since this protective barrier can be disrupted by organic solvents, we assessed whether ethanol co-administration can enhance adenovirus-mediated transgene expression. METHODS: Normal and bladder tumor-bearing rats received a single intracystic administration of rAd encoding beta-galactosidase (rAd-beta gal) or p53 (rAd-p53). rAd was administered in a saline solution or in solutions with increasing concentrations of ethanol. Transgene expression was evaluated in the bladder tissues. RESULTS: A dramatic increase in urothelial beta-galactosidase transgene expression was achieved by rAd-beta gal administered in a 22% ethanol solution. Transgene expression was enhanced in normal urothelium and in superficial bladder tumors. p53 transgene expression was similarly enhanced. CONCLUSIONS: Co-administration of 22% ethanol enhanced local rAd-mediated transgene expression in the normal and neoplastic bladder epithelium in rodents. Improvement of rAd-mediated transgene expression is progress toward local gene delivery to the urothelium and may enable local gene therapy for superficial bladder cancer or other bladder diseases.

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo.

Alterations in the p53 tumor-suppressor gene occur in 35-60% of human glioblastomas, and re-introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication-deficient recombinant adenoviruses encoding wild-type p53 (rAd-p53). Western blot analyses showed high-level expression of p53 protein after treatment with rAd-p53, and transgene expression was dependent on promoter strength. A p53-specific dose-dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus-mediated gene transfer and expression. p53-specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd-p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd-p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re-introduction of wild-type p53 may have potential clinical utility for gene therapy of glioblastoma.

Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene.

We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.

Recombinant human retinoblastoma protein inhibits cancer cell growth.

Aberrant expression of the tumor suppressor gene RB1 is associated with a variety of solid tumors and hematopoietic neoplasms. Certain cancer cell lines in which the protein encoded by RB1 (p110RB) is absent have been reported to show decreased growth rate, clonogenicity, or tumorigenicity following insertion of a transcriptionally active RB1 gene. We asked whether these RB-deficient cells could be growth inhibited by direct exposure to purified p110RB. We report a decrease in uptake of tritiated thymidine by 5637 bladder carcinoma cells (RB-negative) when purified recombinant p110RB is added to culture media. Internalization of the protein by cells and translocation to the nucleus are demonstrated by immunohistochemistry, FACS, and detection of radiolabeled protein in subcellular fractions. Next, we chose a well-described leukemia cell culture model to investigate the potential effect of recombinant p110RB in clinical disease. We observed dose-related decreases in cell number of colony formation in vitro in 8 of 20 acute myelogenous leukemia samples, 7 of which did show endogenous p110RB detectable by immunohistochemistry. Histological appearance following exposure to p110RB shows cytoplasmic vacuolization and nuclear lobulation of degenerating cells. We conclude that purified p110RB added to culture media is internalized by cells, translocated to the nucleus, and exerts a growth-inhibitory effect on certain cancer cell types.

Inhibition of tumor cell proliferation in vitro and in vivo by exogenous p110RB, the retinoblastoma tumor suppressor protein.

Reconstitution of retinoblastoma gene (RB) deficient tumor cells with RB generally leads to growth suppression in vitro and/or reduced tumorigenicity in nude mice. An alternate approach to gene replacement is the delivery of the RB gene product (p110RB) into cells lacking its expression. In this report we demonstrate that exogenously added p110RB is taken up by and localized to the nucleus of cultured cells and has growth suppression properties similar to endogenous RB. RB-negative (RBneg) tumor cells are preferentially growth inhibited while most RB-positive (RBpos) tumor cells and normal cells are much less sensitive. We have extended these studies to relevant nude mouse xenograft models for human lung cancer. Local or systemic administration of p110RB inhibits tumor growth in treated animals. These results represent the first use of a tumor suppressor protein as a potential cancer therapeutic.

Melphalan sensitivity as a function of progressive metastatic growth in two subpopulations of a mouse mammary tumour.

In order to examine in detail the sensitivity to chemotherapy of tumour cells at various organ sites and at various stages of metastasis, we have used a series of cell lines, all selected from sister subpopulations derived from a single mouse mammary tumour, which can be distinguished and quantitated from normal cells and from each other through growth in selective medium. For the studies described here, we used two lines, 4T07 and 66FAR, which will form colonies in vitro in medium containing 60 microM 6-thioguanine or 330 microM 2,6-diaminopurine, respectively. Both cell lines have similar sensitivity to the test chemotherapeutic agent, melphalan, in vitro. These two tumour cell lines were treated with melphalan in vivo, when growing either in lungs as experimental metastases at various times after cell injection or as palpable tumours growing subcutaneously. Responses to various doses of melphalan were measured by removing lungs or subcutaneous tumours and performing colony-forming assays in selective medium. The data indicate marked shifts in sensitivity as a function of metastatic stage. Analyses of dose-response curves show that both cell lines were similarly sensitive to melphalan at early times (45 min) after cell injection i.v. but became less sensitive at an intermediate time after injection (3 days). Differences between the two lines became apparent at later times after i.v. injection (by day 8 or 9) and in subcutaneous tumours, where a marked reduction in the shoulder of the dose response curve was seen in line 4T07, resulting in sensitivity equal to or greater than the of early times, whereas the dose response parameters of 66FAR remained at those of the intermediate time point. These results show that, in heterogeneous tumours, individual subpopulations of tumour cells may respond differently to chemotherapeutic agents at various disease stages. In vitro measures of tumour sensitivity do not predict these changes in in vivo sensitivity. Model systems similar to the one described here may yield information which will eventually be useful in maximising the efficacy of clinically relevant adjuvant chemotherapy regimens.

Tumor subpopulation interactions affecting melphalan sensitivity in palpable mouse mammary tumors.

Paired mixtures of melphalan-sensitive and relatively insensitive tumor cell subpopulation lines, originally derived from the same mammary tumor, were injected s.c. into syngeneic mice. When tumors were palpable, the mice were treated with melphalan at doses shown to be effective against the melphalan-sensitive subpopulations. Sensitivity was assessed by the loss of colony-forming ability of tumor cells harvested 1 to 14 days after treatment. When growing in tumors mixed with melphalan-sensitive line 4TO7 cells, line 66 (less sensitive) appeared much more sensitive than when it was grown alone. Line 66 tumors growing on the opposite sides of mice bearing line 4TO7 tumors were not more sensitive than when grown alone, indicating the lack of a systemic mechanism in the transfer of sensitivity from 4TO7 to 66. Furthermore, line 66 was not more sensitive when mixed with line 168TFAR (another melphalan-sensitive subpopulation) than when alone. The "transfer of sensitivity" from line 4TO7 to line 66 could be reproduced in collagen gel cultures but not in monolayer. Interestingly, line 4TO7, unlike line 168TFAR, is more sensitive to melphalan in collagen culture than in monolayer. This difference in sensitivity does not appear to be influenced by differences in cell density between the two culture systems. In collagen culture, the increased sensitivity of line 66 in the presence of line 4TO7 did not require cell contact and so appeared to act through diffusible factors(s). Collectively, these data suggest that the transfer of sensitivity is not dependent upon host factors or upon drug sensitivity per se but rather upon some mechanism requiring tumor cell-tumor cell interaction between specific subpopulation pairs. In additional studies, pH was ruled out as a factor in the transfer of sensitivity.