No abstract

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.

An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT] gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial f8-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis. (J.

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.

3T3-L1 adipoblasts that express large amounts of c-Myc cannot terminally differentiate, raising the possibility that Myc inhibits the expression of genes that promote adipogenesis. The CCAAT/enhancer binding protein (C/EBP alpha) is induced during 3T3-L1 adipogenesis when cells commit to the differentiation pathway. Transfection of 3T3-L1 adipoblasts with the gene that encodes C/EBP alpha caused overt expression of the adipocyte morphology. Expression of Myc prohibited the normal induction of C/EBP alpha and prevented adipogenesis. Enforced expression of C/EBP alpha overcame the Myc-induced block to differentiation. These results provide a molecular basis for the regulation of adipogenesis and implicate Myc and C/EBP alpha as pivotal controlling elements.

A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, I examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state:(i) the ability to arrest growth in GWG1, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to lose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell lines expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter.Cells that expressed high constitutive levels of pRSVmyc mRNA arrested in GO/G1 at densities siiMilar to those of normal cells at confluence. Upon initiation of the diffekrentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G0/Gl. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G0/G1 or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate. The block to differentiation could be reversed by high expression of myc antisense RNA, showing that the induced block was specifically due to enforced expression of pRSVmyc. These findings indicate that 3T3-L1 preadipocytes enter a specific state in GWG1 after treatment with differentiation inducers, into which cells expressing high constitutive levels of myc RNA are precluded from entering. I propose that myc acts as a molecular switch and directs cells to a pathway that can lead to continued proliferation or to terminal differentiation.

No abstract

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTKSR39rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na99mTcO4 in infected cells using single photon emission–computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 × 1012 virus particles (vp) but not at 1 × 1011 vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 × 1012 vp in 1 cm3, GE volume (GEV) increased to a mean of 6.6 cm3, representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1–2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.