Molecular characterization of the genome of a partitivirus from the basidiomycete Rhizoctonia solani.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungus Rhizoctonia solani isolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5' ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem-loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86-2275; molecular mass 86 kDa) and 683 aa (bases 79-2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses from Fusarium poae and Atkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.

Regulation of differentiation and protein kinase C expression in 3T3 T proadipocytes: effects of TGF-beta and transformation.

We are studying the mechanisms that regulate proliferation and differentiation of normal 3T3 T proadipocytes and neoplastically transformed clones which have lost the ability to differentiate. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and transforming growth factor beta (TGF-beta) are known inhibitors of the same step of the differentiation process in 3T3 T cells. Here, we examined the expression of the phorbol ester receptor/protein kinase C (PKC) during adipocytic differentiation of 3T3 T cells and its modulation by the differentiation inhibitor TGF-beta. PKC receptor assays were performed using a tritiated analogue of TPA and it was found that PKC receptor levels decreased approximately threefold during differentiation. Northern blot analyses revealed an even greater decrease of PKC transcripts during differentiation. TGF-beta inhibited not only differentiation, but the differentiation-dependent decrease in PKC levels as well. Transformed 3T3 T cells which have lost the ability to differentiate were found to express aberrant levels of PKC. The data suggest that TGF-beta may inhibit differentiation via a PKC-dependent pathway and that disruption of normal PKC levels or its regulation may be involved in the loss of differentiation control in transformed 3T3 T cells.

Loss of differentiation control in transformed 3T3 T proadipocytes.

Nontransformed 3T3 T mesenchymal/proadipocyte stem cells can be readily induced to differentiate, yet previous work has shown that 3T3 T cells that are spontaneously or virally transformed not only lose their normal growth control mechanisms but also lose the ability to differentiate. Loss of growth control can be due to autocrine mechanisms in some transformed cells, but the mechanisms involved in disrupting differentiation control are poorly understood. Our goal is to further define the growth and differentiation defects that arise in neoplastically transformed cells and the mechanisms underlying those defects. For example, exogenous transforming growth factor beta and tumor necrosis factor, both of which are secreted aberrantly by some tumor cells, are known inhibitors of different steps of the normal 3T3 T adipocyte differentiation process, suggesting a potential role as autocrine factors in disrupting differentiation of transformed 3T3 T cells. In the current study we transformed 3T3 T cells in vitro with chemical or UV irradiation treatment in order to determine if the acquisition of the transformed phenotype after these treatments is also associated with loss of differentiation control as it is with spontaneously or virally transformed cells. Four chemically and two UV-treated 3T3 T cell lines were isolated from type III foci and all have been found to be tumorigenic in syngeneic animals and to have lost the ability to differentiate. Relative to the parental cell line the differentiation abilities of the transformed clones ranged from 0 to less than 5%. In this regard, we also analyzed the normal and aberrant expression of three growth factors and differentiation inhibitors in transformed cells. Both transforming growth factor alpha and beta were found to be expressed in non-transformed 3T3 T cells as determined by Northern blot analyses. In addition, both were found to be down-regulated during differentiation of 3T3 T cells. Transformed/differentiation-defective 3T3 T cells expressed varied levels of transforming growth factor alpha and beta. Three of the new transformed clones expressed particularly high levels of transforming growth factor alpha. Very low levels of tumor necrosis factor expression were found in the normal cells and the transformed cells appeared to express tumor necrosis factor at similar levels. In contrast, none of the transformed cells expressed any of the differentiation-specific genes tested (lipoprotein lipase, glycerol-3-phosphate dehydrogenase, etc.). Even a transformed clone which could undergo growth arrest but not morphological differentiation expressed no differentiation-specific genes. Together, these data suggest that neoplastic transformation in general disrupts differentiation control.(ABSTRACT TRUNCATED AT 400 WORDS)

TGF-beta blocks early but not late differentiation-specific gene expression and morphologic differentiation of 3T3 T proadipocytes.

Transforming growth factor-beta (TGF-beta) inhibits morphologic differentiation of BALB/c 3T3 T cells as well as other proadipocyte models. Our prior studies suggested that TGF-beta may act only during the early stages of differentiation induction. However, we did not determine whether TGF-beta was differentially effecting expression of any of the various differentiation-specific genes or if it could cause down-regulation of these genes in differentiated cells. Therefore, in the current study we tested the effects of exogenous TGF-beta (0.01-5.0 ng/ml) on morphologic differentiation and on differentiation-dependent gene expression (Northern and slot blot analyses) at various times during differentiation. When induced to differentiate, 3T3 T cells first undergo predifferentiation growth arrest and from this state molecular, biochemical, and morphological differentiation proceeds. Here it was found that when added prior to the onset of differentiation, TGF-beta was a potent inhibitor or morphologic differentiation as well as of the expression of differentiation-specific genes such as lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPD). However, once morphologic differentiation began, TGF-beta was ineffective in blocking differentiation. In addition, exposure of fully differentiated cells to TGF-beta for up to 72 hours caused no decrease of differentiation-specific genes and even a 7-day treatment caused no morphologic dedifferentiation. Tumor necrosis factor also had no detectable effect on fully differentiated cells.

Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells.

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet-poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0-200 micrograms/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose-dependent manner in medium containing 10% FBS once they reached confluence. The data suggest that the ability of AD4743 to inhibit growth may also be coupled with its ability to enhance differentiation. In addition, AD4743 (1-10 micrograms/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (less than 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80-95% differentiation could be achieved in just 4 days, compared to 8-12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to a greater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation-specific genes. AD4743 at 1-10 micrograms/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin-dependent pathway.