Function of an inhibitor of cyclin-dependent kinase p27/Kip in cells transformed by E1A + E1B19 kDa + E1A + cHa-Ras, differing in their ability to realize a G1-block during serum starvation

We studied the capability of E1A + cHa-ras and E1A + E1B19kDa transformants to undergo the G1/S arrest of the cell cycle following depletion of serum growth factors. It has been shown that serum starvation induced the G1/S arrest both in normal rat embryo fibroblasts (REF) and in E1A + E1B19kDa transformants, whereas E1A + cHa-ras transformed cells lost this feature. To analyse the mechanisms underlying these differences, we studied the expression of p27/KIP, its intracellular distribution and association with E1A oncoproducts. The content of the p27/KIP inhibitor of cyclin-dependent kinases was found to change a little upon transformation by two complementary oncogene pairs. However, serum starvation for 24 h led to a significant increase in the content of p27/KIP in E1A + E1B19kDa transformants, while E1A + cHa-ras cells accumulated p27/KIP less markedly. According to the immunofluorescence study, the p27/KIP inhibitor is located in the nucleus of both normal and transformed cells. Moreover, serum starvation did not lead to its inhibition due to redistribution to the cytoplasm in both cell lines. Also, we were unable to detect association of p27/KIP with E1A oncoproducts in immunoprecipitated complexes. The obtained data indicate that, in contrast to E1A + cHa-ras transformants, in E1A + E1B19kDa cells the p27/KIP inhibitor is functional and it is capable of inducing the G1/S block after serum starvation.     

Specific regulated endonuclease activity of small RNP, containing prosomes from the A431 cell line: possible mechanisms of regulating the stability of RNA by epidermal growth factor

A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.     

Cyclin D2 and Ha-Ras transformed rat embryo fibroblasts exhibit a novel deregulation of cell size control and early S phase arrest in low serum.

The D-type cyclins are growth factor-regulated delayed early functions which peak at the G1/S transition, are thought to regulate entry into S phase and have been implicated in tumorigenesis. Here, we show that cyclin D2 can co-operate with Ha-Ras to impose a novel transformed state on rat embryo fibroblasts (REF). While clonal cyclin D2/Ha-Ras REF transformants exhibit a characteristic transformed phenotype in high serum, in low serum they arrest cell proliferation and display profound morphological and cytological changes indicating loss of control of cell mass and deregulation of the G1/S transition. Notably, in low serum, despite re-establishment of actin cables and arrest of proliferation, cell mass continues to increase, creating giant cells up to 10 x normal size. Also, during low-serum culture the cells make a very gradual but progressive entry into S phase, reaching a 2.4N DNA content after 6 days. PCNA is expressed and 2N and 4N cells are largely absent, and thus the cells undergo a novel S phase arrest. While transfer to low serum induced the retinoblastoma protein to enter its dephosphorylated state, and cyclin A, cyclin B and cdc2 levels to decrease, all as normal, cyclin E, cdk4, cdk2 and the exogenous cyclin D2 persisted at high levels. These results indicate that cyclin D2 and Ha-Ras can transform cells when mitogenic signals from growth factors are provided. However, in low serum, co-operation of cyclin D2 and Ha-Ras provides only a subset of the progression signals and these are sufficient for G1-related cell mass increase and S phase entry, but are insufficient for full cell cycling.     

Rat embryo fibroblasts transformed by complementation with oncogenes E1A+E1B-19 and E1A+cHa-ras differ in the ability to realize the G1/S block in serum free media

The capability of REF cells transformed by EA + E1B-19 kDa and EA + cHa-ras oncogenes to realize the G1/S cell cycle arrest upon serum starvation was studied. The amount of cyclin-kinase inhibitor protein p27/Kip was shown to increase in both normal and transformed cells. However, the p27/Kip-bound cyclin-kinase complexes of transformed cells were found to be active, implying the functional inactivation of p27/Kip inhibitor. Nevertheless, in contrast to E1A + cHa-ras transformants, E1A + E1B-19 kDa transformants undergo the G1 cell cycle arrest. The G1 cell cycle block correlates with the decrease in cyclinE-Cdk2 activity. Since cyclinE-Cdk2 complexes need Thr-160 phosphorylation of Cdk2 by CAK-kinase for full activity, we have analysed the Cdk-7 associated activity upon serum starvation using gst-Cdk2 as a substrate. Serum starvation did not affect CAK activity either in E1A + cHa-ras or in E1A + E1B-19 kDa transformants. Thus, selective suppression of cyclineE-Cdk2 activity in E1A + E1B-19 kDa transformants upon serum starvation does not arise from the action of cyclin-kinase inhibitors, or from change in CAK activity.     

Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9.

Interleukin-9 (IL-9) activates three distinct STAT proteins: STAT1, STAT3, and STAT5. This process depends on one tyrosine of the IL-9 receptor, which is necessary for proliferation, gene induction, and inhibition of apoptosis induced by glucocorticoids. By introduction of point mutations in amino acids surrounding this tyrosine, we obtained receptors that activated either STAT5 alone or both STAT1 and STAT3, thus providing us with the possibility to study the respective roles of these factors in the biological activities of IL-9. Both mutant receptors were able to prevent apoptosis, but only the mutant that activated STAT1 and STAT3 was able to support induction of granzyme A and L-selectin. In line with these results, constitutively activated STAT5 blocked glucocorticoid-induced apoptosis. In Ba/F3 cells, significant proliferation and pim-1 induction were observed with both STAT-restricted mutants, though proliferation was lower than with the wild-type receptor. These results suggest that survival and cell growth are redundantly controlled by multiple STAT factors, whereas differentiation gene induction is more specifically correlated with individual STAT activation by IL-9.