E1A-transformed Cells Research Articles

Suppression of the p300-dependent mdm2 negative-feedback loop induces the p53 apoptotic function.

The p53 tumor suppressor gene product interacts with the p300 transcriptional coactivator that regulates the transactivation of p53-inducible genes. The adenovirus E1A protein has been shown to bind to p300 and inhibit its function. E1A inhibits p53 transactivation and also promotes p53 accumulation by a p300-dependent mechanism. Murine double minute 2 (Mdm2) is a transcriptional target of p53 that binds to p53 and inhibits its transcriptional activity. E1A inhibited mdm2 transactivation without affecting the expression of p21(WAF1) or Bax, which resulted in high levels of p53 accumulation and apoptosis. Ectopic expression of p300 restored Mdm2 levels and inhibited p53-dependent apoptosis, as did ectopic expression of Mdm2. Thus, p300 is required for mdm2 induction by p53 and the subsequent inhibition of p53 stabilization. Inhibition of p300 by E1A results in stabilization of p53 and causes apoptosis. Moreover, E1B 19K or Bcl-2 expression in E1A-transformed cells abrogated p53-dependent apoptosis by restoring mdm2 transactivation by p53. Hence, p300 regulation of mdm2 expression controls apoptotic activity of p53, and 19K or Bcl-2 bypass E1A inhibition of p300 transactivation of Mdm2.

Mechanism of E1A-Induced Transforming Growth Factor-β (TGF-β) Resistance in Mouse Keratinocytes Involves Repression of TGF-β Type II Receptor Transcription

Cellular transformation driven by the E1A oncogene is associated with the development of cellular resistance to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). We demonstrate that development of resistance occurs simultaneously with decreased expression of TGF-beta type II receptor (TGF-beta RII) mRNA and protein. To determine whether changes in transcriptional regulation are responsible for the decreased receptor expression in E1A-transformed cells, a series of mobility shift assays was performed utilizing nuclear extracts from E1A-transformed and untransformed murine keratinocytes using radiolabeled positive regulatory elements (PRE1 and PRE2) of the TGF-beta RII promoter. The results from these assays suggest that E1A-transformed cells express markedly lower levels of nuclear proteins that bind specifically to PRE1 and PRE2. Transfection of both E1A-transformed and untransformed cell lines with a series of mutant promoter constructs confirmed that both PREs contribute significantly to basal expression of TGF-beta RII and that inactivation of either element leads to markedly reduced promoter activity. We conclude that development of TGF-beta resistance in E1A-transformed cells is achieved in part through transcriptional down-regulation of the TGF-beta RII gene and that this down-regulation is the result of decreased expression of unidentified transcription factor complexes that interact with PRE1 and PRE2.

Modulation of cellular chemoresistance in keratinocytes by activation of different oncogenes.

Response to chemotherapeutic agents in malignant tumors depends on many factors, most of which are as yet unknown. We investigated the correlation between the activation of different oncogenes and protein-kinase-C (PKC) modulation, and the cytotoxicity of some of the most widely used anti-cancer drugs. We transformed the murine keratinocyte cell line PAM 212, with different oncogenes (v-H-ras, v-myc and adenovirus E1a) and a mutant p53 suppressor gene (mp53). The cytotoxic effect of cisplatin (CDDP), doxorubicin (DOX) and vincristine (VCR), together with the concomitant action of modulators of PKC, TPA and staurosporine were evaluated by the crystal-violet method, thymidine incorporation and flow cytometry. We report that (a) the oncogene v-H-ras induces resistance to CDDP (> 50%), DOX (> 25%) and VCR (> 20%); (b) the E1a oncogene induces only resistance to VCR (> 40%) and marked sensitivity to CDDP and DOX; (c) the mp53 oncogene induces more resistance to VCR and insignificant resistance to the other drugs; and (d) activation of PKC by TPA increases the resistance to VCR and DOX in cells transformed by the v-H-ras, while it significantly increases the lethality with CDDP of the E1a-transformed cells. Staurosporine increases the cytoxicity of all the drugs, especially in the E1a-transformed keratinocytes. In the flow-cytometry analysis, the percentage of BUdR incorporation was related to sensitivity to anti-cancer drugs.

Counteracting Effects of E1a Transformation on cAMP Growth Inhibition

Several signaling molecules have been identified which act as inhibitors of epithelial cell growth. The mechanisms for this negative growth regulation are still poorly understood. In the case of TGF-β, inhibition of keratinocyte cell growth can be totally prevented by transformation with an intact early region 1a (E1a) oncogene. We show here that E1a-transformed keratinocytes become also partially resistant to growth inhibition by elevated 3′,5′-cyclic adenosine monophosphate (cAMP) levels, as induced by treatment with forskolin, dibutyryl-cAMP, 8Br-cAMP, or 8C1-cAMP. Resistance to cAMP is due to interference of E1a with signaling pathways downstream of protein kinase A (PKA) activation, as intracellular cAMP levels and PKA activity were found to be similar in control and E1a-transformed cells. Induction of c-fos expression by 8Br-cAMP occurs at the same time in both cell lines. Interestingly however, this effect is maintained longer in the case of E1a-transformed cells compared to the control. A truncated E1a mutant which is still able to bind to the p105-Rb gene product, p107, and p60/cyclin A, induces cAMP resistance at levels which are only slightly lower than those induced by an intact E1a oncogene. In contrast, an E1a mutant which binds only to a p300 cellular protein and induces a substantial level of TGF-β resistance fails to induce cAMP resistance. Thus, E1a transformation counteracts the growth-inhibitory effects of cAMP as well as TGF-β, but to a different degree and through an only partially overlapping mechanism.

Glucocorticoid hormones may partially substitute for adenovirus E1A in cooperation with ras

The effects of hormonal promotion of T24- ras oncogene-transfected rat embryo fibroblasts (REF) were compared to cotransformation of these cells with adenovirus E1A and ras. Cotransfection of E1A + ras resulted in the appearance of morphologically transformed cells which were very efficiently established into cell lines. Addition of glucocorticoid hormones to T24- ras-transfected REF cells resulted in cells with a transformed morphology and a capacity to form foci. These foci were, however, inefficiently established into stable cell lines. Removal of hormone from growing cells resulted in retarded growth, suggesting that the hormone acted as a growth factor on these cells. Both E1A-transformed cells and hormone-treated ras- transformed cells showed a reduction in synthesis of high molecular weight tropomyosin isoforms and a decreased expression of surface fibronectin. Control experiments demonstrated that the effects of hormone were mediated through the glucocorticoid receptor. Our findings suggest that glucocorticoid hormones may promote the in vitro growth of ras-initiated REF cells into stably transformed cell lines, but that this ability is limited compared to that of adenovirus E1A.

Induction of transforming growth factor beta 1 resistance by the E1A oncogene requires binding to a specific set of cellular proteins.

Transforming growth factors beta (TGF-beta s) are potent inhibitors of epithelial cell growth in culture and might play a similar role in vivo. Several studies have suggested that acquisition of TGF-beta resistance is an important step in epithelial tumor development. Here, we show that resistance to TGF-beta 1 growth inhibition can be induced by transformation of keratinocytes with the E1A, but not the ras, oncogene. Mutational analysis revealed that these effects closely correlate with the ability of E1A proteins to bind to the retinoblastoma gene product (p105) as well as to three other cellular proteins (p60, p107, and p300). Only partial resistance to TGF-beta 1 growth inhibition was elicited by E1A mutants that bind to a subset of proteins, whereas complete resistance was induced by E1A mutants that bind to all four proteins together. Total protection against TGF-beta growth inhibition was also induced by concomitant introduction into cells of an E1A mutant binding to the p60/p105/p107 proteins and one binding to p300. In parallel with these effects, epidermal transglutaminase, a marker of keratinocyte differentiation, was induced by TGF-beta in control but not in E1A-transformed cells. TGF-beta 1 receptor levels were only partially down-modulated by an intact E1A gene and not significantly affected by the various truncated mutants. Thus, the ability of E1A to induce TGF-beta resistance depends on its ability to bind, and presumably inactivate, several cellular proteins that may be involved in transmission of the TGF-beta signal and seem to act downstream from its receptor(s).

Suppression of block to entry into S phase in cell-cycle mutants of rat 3Y1 fibroblasts after transformation by adenovirus type 12

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, belonging to separate complementation groups, cease to proliferate in the G1 phase of the cell cycle at a restrictive temperature (39.8°). These is mutants were transformed at a permissive temperature with adenovirus type 12 (Ad12), its E1 A gene, or in203S mutant of Ad12 which has a mutation in the E1 A 13 S mRNA unique region. We examined whether the proliferation of the transformed cells would be blocked in the G1 phase, at 39.8°. One mutant did not cease to proliferate at 39.8° after transformation with either Ad1 2 or E1 A. In two other mutants, Ad12-transformed cells did not cease to proliferate at 39.8°, whereas E1A-transformed cells did not survive at 39.8°, though they did continue to enter the S phase. Analysis of transcription of the viral early genes in the transformants of one of the latter two mutants suggests that the expression of other viral early genes, in addition to E1 A, is required for cell proliferation, in addition to entry into S phase. In the fourth mutant, both Ad12- and E1A-transformed sublines did not cease to enter the S phase but cells readily detached from the dishes. These results suggest that (1)function(s)of the E1A gene alone is sufficient for Ad12 to suppress the inhibition of the initiation of cellular DNA synthesis caused by four different cellular is defects, (2) functions of Ad12 early genes other than, or in addition to, E1 A are necessary for suppression of the inhibition of cell proliferation (and/or for survival) in two of the four is mutants, and (3) in the case of one other is mutant, E1 A alone overcomes the is defect and allows for the entire cell proliferation.

Protein synthesis in adenovirus E1A transformed cells.

Protein synthesis in cells transformed by the E1A gene of adenovirus type 12 (Ad12) was investigated using rat fibroblast cells (3Y1), these cells transformed by the E1A gene of Ad 12 (HY1), human epithelial carcinoma cells (KB) and their transformant (KB-E1A), and mouse mammary tumor cells (FM3A) and their transformant (FM3A-E1A). The proteins labeled with (32S)-methionine in the intact cells or synthesized using an in vitro translation system were analyzed by two dimensional gel electrophoresis.The E1A gene of Ad 12 increased the number of protein spots in the gel. On the other hand, TPA (12-O-tetradecanoyl-phorbol-13-acetate) increased the number of protein spots in 3Y1 cells while TPA did not seem to have the same effect in HY1 cells. In vitro translation of the mRNAs from 3Y1, HY1, KB, and KB-E1A cells have shown that the number of protein spots in the gel were greater in the E1A-transformed cells than non-transformed cells.FM3A cells transformed by the E1A gene of Ad12 drastically suppressed their i...