Activation of the cyclin D2 and cdk6 genes through NF-kappaB is critical for cell-cycle progression induced by HTLV-I Tax.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of cell types, so the cause of T-cell-specific oncogenesis remains to be elucidated. The trans-activator protein Tax of HTLV-I can promote cell-cycle progression in resting T cells along with induction of cyclin D2 and cyclin-dependent kinase (cdk6) gene expression. Here, we found that Tax cannot induce cell-cycle progression in resting fibroblasts and analysed the molecular basis of the cell-type specificity. Tax activated cyclin D2 and cdk6 promoters in T cells, but not in fibroblasts, depending on its ability to activate the transcription factor nuclear factor (NF)-kappaB. Expression of cyclin D2 and CDK6 activated the transcription factor E2F, which is essential for cell-cycle progression, in both T cells and fibroblasts. Short-hairpin RNA (shRNA)-mediated inhibition of cyclin D2 and CDK6 induction suppressed Tax-induced activation of E2F in T cells. Finally, shRNA-mediated downregulation of NF-kappaB p65 or p100 expression reduced Tax-induced activation of cyclin D2 and/or cdk6 promoters and cell-cycle progression in T cells. These results indicate that Tax-induced cell-cycle progression in T cells is mediated, at least in part, through cell-type-specific activation of the cyclin D2 and cdk6 genes through NF-kappaB and may be important for the cell-type-specific oncogenesis.

Defect in serine 46 phosphorylation of p53 contributes to acquisition of p53 resistance in oral squamous cell carcinoma cells.

To investigate whether dysregulation of p53 phosphorylation confers tumor resistance to p53, we analysed the effects of wild-type p53 on oral squamous cell carcinoma (SCC) cell lines carrying various mutations of p53. Introduction of exogenous p53 neither induced apoptosis nor suppressed colony formation in HSC-3 cells lacking any detectable p53 and HSC-4 cells expressing mutant p53R248Q protein. Consistently, exogenous p53 did not induce proapoptotic p53-target genes in these p53-resistant cells. We found that phosphorylation of exogenous p53 on serine 46 (Ser46) was severely impaired in HSC-3 but not HSC-4 cells. A mutant mimicking Ser46-phosphorylation (p53S46D) enhanced proapoptotic Noxa promoter activity, and overcame the resistance to p53-mediated apoptosis and growth suppression in HSC-3 cells. Conversely, a mutant defective for Ser46-phosphorylation (p53S46A) failed to suppress the growth of p53-sensitive HSC-2 cells. In contrast to HSC-3 cells, p53S46D had no effect on HSC-4 cells, and inhibition of endogenous p53R248Q by siRNA restored p53-mediated apoptosis in HSC-4 cells, indicating a dominant-negative effect of p53R248Q protein on wild-type p53 function. These results demonstrate that the defect in Ser46 phosphorylation accounts for the p53 resistance of HSC-3 cells, and provide evidence for a mechanism underlying the acquisition of p53 resistance in oral SCC.

Neonatal estrogen exposure inhibits steroidogenesis in the developing rat ovary.

Treatment of newborn female rats with estrogens significantly inhibits the growth and differentiation of the ovary. To understand the molecular mechanism of estrogen action in the induction of abnormal ovary, we examined the expression profiles of steroidogenic factor 1 (SF-1) and several of its target genes in the developing ovaries after neonatal exposure to synthetic estrogen, estradiol benzoate (EB) by using reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. Morphologic examination indicated inhibitory effects of estrogen on the stratification of follicles and development of theca and interstitial gland during postnatal ovarian differentiation. The expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450(SCC)), which are both essential for steroid biosynthesis, markedly decreased in theca and interstitial cells throughout the postnatal development of the EB-treated ovary. However, expression of the transcriptional activator of the two genes, SF-1 was unaffected in theca and interstitial cells, although the number of these cells was lower in the EB-treated ovary than in the control ovary. The expression of the estrogen mediator, estrogen receptor-alpha (ER-alpha), diminished specifically in theca cells at P6 and recovered by P14 in the EB-treated ovary. These results indicate that the effect of estrogens is mediated by means of ER-alpha resulting in the down-regulation of StAR and P450(SCC) genes during early postnatal development of the ovary. These results suggest that the abnormal ovarian development by neonatal estrogen treatment is closely correlated with the reduced steroidogenic activity, and the data obtained by using this animal model may account in part the mechanism for aberrant development and function of the ovary in prenatally estrogen-exposed humans.

Essential roles for G1 cyclin-dependent kinase activity in development of cardiomyocyte hypertrophy.

Although cardiomyocytes undergo terminal differentiation soon after birth, irreversibly withdrawing from the cell cycle, growth stimulation induces cell hypertrophy. Such growth stimulation is also responsible for the upregulation of G1 cyclins and cyclin-dependent kinase (CDK) activity in proliferating cells. We sought to determine whether G1 CDK activity is involved in the hypertrophy of rat neonatal cardiomyocytes in culture. We show that serum stimulation promoted the G1 CDK activity without induction of DNA synthesis in cardiomyocytes. Furthermore, overexpression of CDK inhibitors p16(INK4a) and p21(CIP1/WAF1) by use of the adenovirus vector effectively prevented cell enlargement and depressed serum-induced protein synthesis and expression of skeletal alpha-actin and atrial natriuretic factor, genetic markers of cardiac hypertrophy. These results suggest that the G1 CDK activity promoted by serum stimulation is required for the induction of cardiomyocyte hypertrophy and provide novel evidence for understanding the regulation of cardiac hypertrophy by cell cycle regulators.

A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.

Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

Identification of distinct roles for separate E1A domains in disruption of E2F complexes.

The adenovirus E1A protein can disrupt protein complexes containing the E2F transcription factor in association with cellular regulatory proteins such as the retinoblastoma gene product (Rb) and the Rb-related p107 protein. Previous experiments have shown that the CR1 and CR2 domains of E1A are required for this activity. We now demonstrate that the CR2 domain is essential for allowing E1A to interact with the E2F-Rb or the E2F-p107-cyclin A-cdk2 complex. Multimeric complexes containing E1A can be detected when the CR1 domain has been rendered inactive by mutation. In addition, the E1A CR1 domain, but not the CR2 domain, is sufficient to prevent the interaction of E2F with Rb or p107. On the basis of these results, we suggest a model whereby the CR2 domain brings E1A to the E2F complexes and then, upon a normal equilibrium dissociation of Rb or p107 from E2F, the E1A CR1 domain is able to block the site of interaction on Rb or p107, thereby preventing the re-formation of the complexes.