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 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.

Adenovirus-mediated gene transfer may hold much promise in the treatment of human cancer. However, concerns regarding vector dissemination beyond the target tissue, particularly with replication-competent viruses, require an evaluation of the persistence of viral infection in collateral tissue and vector-associated toxicities. In addition, for indications such as prostate cancer, the proximity of the point of viral administration to organs of the male reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus. To address these concerns, the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent adenovirus (Ad5-CD/TKrep) following intraprostatic administration in the mouse was examined. Ad5-CD/TKrep (10(10) vp, 5 x 10(11) vp/kg) was injected intraprostatically on Day 1 of the study and its presence in the major organs of the male urogenital tract (prostate, testes, seminal vesicles, and urinary bladder) and liver was determined on Days 8 and 29. For comparison, a parallel group of animals was injected with the same dose of a related replication-defective Ad5-FGNR virus. To evaluate germ-line transmission, Ad5-CD/TKrep-injected males were mated to females on Days 8 and 29 and resulting embryos were examined for AdS-CD/TKrep viral DNA. Ad5-CD/TKrep viral DNA was detected in all major organs of the adult male urogenital tract and liver 7 and 28 Days postinjection. Interestingly, relative to the replication-defective Ad5-FGNR adenovirus, the replication-competent Ad5-CD/TKrep virus accumulated to a much greater level (approximately 300-fold) and persisted for a longer period of time in prostate, testes, and liver. This difference could not be explained on the basis of differences in viral infectivity, suggesting that the AdS-CD/TKrep virus may be capable of replicating in mouse tissues in vivo. In vitro infection of six mouse cell lines representing prostate, testes, and liver demonstrated that the Ad5-CD/TKrep virus was indeed capable of replicating in these mouse cell types, albeit with reduced efficiencies relative to human cells. Despite the fact that the Ad5-CD/TKrep vector persisted in the adult male gonads and may have replicated in vivo, we observed no evidence of germ-line transmission in 149 offspring examined. To evaluate the toxicity of combining Ad5-CD/TKrep viral therapy with CD/5-FC and HSV-1 TK/GCV suicide gene therapies as a prerequisite for a human trial, an escalating dose (10(8), 10(9), 10(10) vp) of Ad5-CD/TKrep was administered intraprostatically followed by 7 days of 5-FC and GCV double prodrug therapy. Although the virus persisted in the mouse urogenital tract and liver for up to 28 days postinjection, most of the toxicities observed were expected, minimal, and self-limiting. These results lead us to believe that intraprostatic administration of the Ad5-CD/TKrep virus to humans concomitant with double suicide gene therapy will be associated with acceptable toxicities and will not result in ve...

Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein(CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBP␤ in naïve NIH3T3 cells, and their ability to inhibit C/EBP␤-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBP␤ and inhibit C/EBP␤ DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPAR␥2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBP␤-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBP␤ from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.Adipogenesis is a process in which an undifferentiated mesenchymal cell capable of proliferation matures into a post mitotic, fat-laden adipocyte. This differentiation process results in dramatic changes in gene expression and a spectacular alteration in cell morphology. The early phase of adipocyte differentiation is accompanied by the induction of transcription factors that promote cell cycle withdrawal and activation of cellspecific genes, two key aspects of terminal cell differentiation.CCAAT/enhancer binding protein ␣ (C/EBP␣), 1 a basicleucine zipper protein, is among the transcription factors that are induced prior to the onset of morphological differentiation. C/EBP␣ was the first regulatory protein demonstrated to play a central role in promotion of the adipogenic program. We (1, 2) and others (3-6) demonstrated that C/EBP␣ was both necessary and sufficient to promote adipogenesis in fibroblastic cells such as NIH3T3 and 3T3-L1. These observations, coupled with the fact that mice deficient in C/EBP␣ completely lack mature white or brown adipose tissue (7), demonstrate unequivocally the pivotal role of this transcription factor in adipoge...