The CCAAT/enhancer-binding protein ot (C/EBPa) has been implicated in the regulation of adipoblast differentiation. In this study we investigate the potential of C/EBPot to promote the adipogenic program in a variety of fibroblastic cells. Transduction of the C/EBPc~ gene into eight mouse fibroblastic cell lines by retroviruses and DNA transfection generates adipocyte colonies at variable frequencies. The most dramatic results are obtained with NIH-3T3 cells, in which the percentage of G418-resistant colonies that exhibit the adipocyte morphology is reproducibly >50% when the C/EBPc~ gene is transduced by retroviruses. The ability to promote the adipogenic program requires the potent transcriptional activation domain of C/EBPa and is not observed with C/EBPI3. Paradoxically, in spite of its antimitogenic effects, clonal cell lines that stably express high amounts of C/EBPa can readily be generated. Stable expression of C/EBPot in BALB/c-3T3 cells dramatically enhances their ability to terminally differentiate into adipocytes. The results demonstrate that C/EBPa can efficiently promote the adipogenic program in a variety of mouse fibroblastic cells, including those that have little or no spontaneous capacity to undergo adipogenesis.

Two obstacles limiting the efficacy of nearly all cancer gene therapy trials are low gene transduction efficiencies and the lack of tumor specificity. Recently, a replication-competent, E1B-attenuated adenovirus (ONYX-015) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to improve both the efficacy and safety of this approach, we constructed a similar adenovirus (FGR) containing a cytosine deaminase (CD)/herpes simplex virus type-1 thymidine kinase (HSV-1 TK) fusion gene, thereby allowing for the utilization of double-suicide gene therapy, which has previously been demonstrated to produce significant antitumor effects and potentiate the therapeutic effects of radiation. The FGR virus exhibited the same tumor cell specificity and replication kinetics as the ONYX-015 virus in vitro. Importantly, both the CD/5-FC and HSV-1 TK/GCV suicide gene systems markedly enhanced the tumor cell-specific cytopathic effect of the virus, and, as expected, sensitized tumor cells to radiation. By contrast, neither the FGR virus nor either suicide gene system showed significant toxicity to normal human cells. Both suicide gene systems could be used to suppress viral replication effectively, thereby providing a means to control viral spread. The results support the thesis that the three-pronged approach of viral therapy, suicide gene therapy, and radiotherapy may represent a powerful and safe means of selectively destroying tumor cells in vivo.

Replication-competent adenovirus-mediated suicide gene therapy is an investigational cancer treatment that combines the oncolytic actions of human adenoviruses with the cytotoxic effects of chemo-radiosensitizing genes. Previously, we reported the short-term effects of this therapy in men with local recurrence of prostate cancer after definitive radiotherapy. With a median prostate-specific antigen (PSA) follow-up of 5 years, we report here the effect of the gene therapy on prostate-specific antigen doubling time (PSADT), a surrogate end point with significant prognostic power. When considering all evaluable subjects, the PSADT increased following the gene therapy from a mean of 17 to 31 months (median 16 to 22 months) (P=0.014). Assuming that salvage androgen suppression therapy androgen suppression therapy (AST) was uniformly initiated at a PSA of 15 ng/mL, the gene therapy would have delayed the projected onset of salvage therapy by an average of 2 years. The results indicate that replication-competent adenovirus-mediated suicide gene therapy may provide a potential long-term benefit to patients, as shown by a lengthening of the PSADT, and delay in when salvage therapy is indicated. Given the high morbidity associated with AST, we believe this approach could provide an attractive treatment option for selection of patients experiencing PSA relapse following definitive therapy.

Replication-competent adenoviruses may provide a highly efficient means of delivering therapeutic genes to tumors. Previously, we evaluated in vitro a replication-competent adenovirus (Ad5-CD/TKrep) containing a cytosine deaminase (CD)/herpes simplex type 1 thymidine kinase (HSV-1 TK) fusion gene that allows lytic viral therapy to be combined with double suicide gene therapy. Both the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems enhanced the tumor cell-specific cytopathic effects of the Ad5-CD/TKrep virus in vitro and sensitized cells to radiation. To extend these in vitro findings in vivo, we evaluated the antitumor activity of the Ad5-CD/TKrep virus in combination with double prodrug therapy and radiation therapy. The Ad5-CD/TKrep virus independently demonstrated significant antitumor activity against C33A cervical carcinoma xenografts. Therapeutic outcome was dramatically improved with systemic administration of double, but not single, prodrug (5-FC + GCV) therapy. When used in a neoadjuvant setting, Ad5-CD/TKrep-mediated double suicide gene therapy dramatically potentiated the effectiveness of radiation therapy. The trimodal approach of Ad5-CD/TKrep viral, double suicide gene, and radiotherapies produced significant tumor regression and ultimately 100% tumor cure. The results demonstrate the high therapeutic potential of the trimodal approach and provide a solid foundation for future clinical trials.

Replication-competent adenovirus-mediated suicide gene therapy has proven to be safe in humans when delivered intraprostatically. Although signs of efficacy are emerging, it is likely that further improvements will be needed before this technology will have widespread applicability in the clinic. Toward this end, we have developed a second-generation, replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-ADP) containing an improved yeast cytosine deaminase (yCD)/mutant(SR39) herpes simplex virus thymidine kinase fusion (yCD/mutTK(SR39)) gene and the adenovirus death protein (ADP) gene. Relative to the first-generation Ad5-CD/TKrep adenovirus, Ad5-yCD/mutTK(SR39)rep-ADP demonstrated greater tumor cell kill in vitro and significantly greater tumor control in preclinical models of human cancer. Quantification of transgene volume following direct injection of fadenovirus into human tumor xenografts and the naïve canine prostate demonstrated that ADP enhanced adenoviral spread in vivo. Toxicology studies were performed to determine whether the improved yCD/mutTK(SR39) fusion and ADP genes increased toxicity. Intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-ADP did not result in significantly increased toxicity relative to the parental Ad5-CD/TKrep adenovirus, the latter of which has proven to be safe in two Phase I prostate cancer clinical trials. Together, these results provide the scientific basis for evaluating the safety and efficacy of the second-generation Ad5-yCD/mutTK(SR39)rep-ADP adenovirus in humans.