Transfection with the E1A and E1B-19kDa Oncogenes Does Not Prevent Rat Embryo Fibroblasts from Cell-Cycle Arrest after γ-Irradiation

Introduction of the E1A early region of the human adenovirus type 5 impairs the ability of mammalian cells to stop in the cell cycle at G1/S after damage. Two-parameter fluorescence cell sorting with iododeoxyuridine revealed the radiation-induced G1/S arrest in rat embryo fibroblasts transformed with the complementing E1A and E1B-19kDa oncogenes. This was due to selective inhibition of CyclE/Cdk2-associated kinase activity, while activities of type 2 kinase and of CyclA/Cdk2 complexes remained unchanged. The inhibitor of G1-phase cyclin kinases, p21/Waf1, was accumulated and interacted with target kinases both in normal and in transformed cells after irradiation. As shown by immunoprecipitation, p21/Waf1 formed complexes with the E1A oncoproducts in the transformants, which possibly accounted for its functional inactivation. Kinase modification in cyclin–kinase complexes was assumed to play a key role in regulation of cyclin-dependent kinases in the transformants with inactivated p21/Waf1.     

Analysis of transient G1/S arrest in E1A+E1B-19kDa transformed cells after ionizing radiation

Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.     

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.     

Apoptotic cell death in rat embryo fibroblasts transformed by EiA + cHa-RAS oncogenes after gamma irradiation

A mechanism of apoptotic death of normal rat embryo fibroblasts and of those transformed by E1A + cHa-Ras oncogenes following gamma irradiation has been investigated. The E1A + cHa-Ras transformed cells were shown to express wild type p53 which was able to trans-activate a reporter pG13-luc Plasmid. As a result of trans-activation, an accumulation of universal inhibitor of cyclin-dependent kinases--p21/Waf1 protein and an increase in the proportion of p21/Waf1 expressing cells were observed, The accumulated p21/Waf1 was found to bind with PCNA. The association with PCNA, however, did not lead to suppression of DNA replication according to the data of iododeoxyuridine (IdUr) incorporation. A high proportion of S-phase cells, in combination with cell cycle blocking in G2-phase, promoted polyploidization of E1A + cHa-Ras transformed cells after gamma irradiation. The polyploidic cells with DNA content equal and higher than 8c die 48-72 h following irradiation due to apoptosis. A significant proportion of E1A + cHa-Ras cells with incorporated IdUr contains labeled micronuclei, the fact being a morphological evidence of apoptosis of cells in S-phase of the cell cycle.     

Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death

The tumor suppressor protein p53 is central to the cellular stress response and may be a predictive biomarker for cancer treatments. Upon stress, wildtype p53 accumulates in the nucleus where it enforces cellular responses, including cell cycle arrest and cell death. p53 is so dominant in its effects, that p53 enforcement – or – restoration therapy is being studied for anti-cancer therapy. Two mechanistically distinct small molecules that act via p53 are the selective inhibitor of nuclear export, selinexor, and MDM2 inhibitor, nutlin-3a. Here, individual cells are studied to define cell cycle response signatures, which captures the variability of responses and includes the impact of loss of p53 expression on cell fates. The individual responses are then used to build the population level response. Matched cell lines with and without p53 expression indicate that while loss-of-function results in altered cell cycle signatures to selinexor treatment, it does not diminish overall cell loss. On the contrary, response to single-agent nutlin-3a shows a strong p53-dependence. Upon treatment with both selinexor and nutlin-3a there are combination effects in at least some cell lines – even when p53 is absent. Collectively, the findings indicate that p53 does act downstream of selinexor and nutlin-3a, and that p53 expression is dispensable for selinexor to cause cell death, but nutlin-3a response is more p53-dependent. Thus, TP53 disruption and lack of expression may not predict poor cell response to selinexor, and selinexor’s mechanism of action potentially provides for strong efficacy regardless of p53 function.     

Inhibition of poly(ADP-ribosyl)ation does not prevent lymphocyte entry into the cell cycle

The enzyme poly(ADP-ribosyl)transferase (ADPRT) becomes activated soon after a mitogenic stimulus is applied to lymphocyte cultures. It has also been reported that ADPRT inhibitors prevent cell proliferation when added to cultures at the same time as the mitogen. While this has been ascribed to the need to seal physiologically present DNA strand breaks before cells enter S phase, the presence of DNA strand breaks in quiescent human lymphocytes has been recently questioned. We demonstrate here that non-toxic concentrations of ADPRT inhibitors do not affect lymphocyte blastization and proliferation, as measured by thymidine incorporation and cytofluorimetry. We therefore suggest that ADPRT activation is required for late functions which are not needed for cell cycle progression.     

G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 microM) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27(KIP1) protein when treated with PD 158780 and increased association of p27(KIP1) with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB.     

Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle

A cell-free system assay involving cell freeze-thawing and protein fractionation by ammonium sulfate precipitation was developed to characterize a cytosol binding protein specific for oxysterols in rat embryo fibroblasts. This protein shared common characteristics with the oxysterol-binding protein described in L cells and in normal human lymphocytes: 8 S sedimentation coefficient, sterol-protein complex of Mr 160 600, saturability, high affinity (Kd in the range of 10(-9) M) and specificity for cholesterol derivatives oxidized on the side chain. These compounds were better inhibitors of DNA synthesis than the compounds oxidized on the nucleus, whereas no difference was found between sterols oxygenated either on the side chain or on the nucleus, as far as inhibition of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) was concerned. Macromolecular components capable of specifically binding 25-hydroxycholesterol were also detected in the fibroblast nucleus. The cytosol oxysterol-binding protein showed a sharp increase (5-fold) in the G2M phase of the cell cycle and in exponentially growing cells (maximal binding site number/cell: 43 500, versus 8850 in confluent cells). Neither the affinity nor the sedimentation coefficient of the protein changed in either situation. The quantitative (but not qualitative) variations of oxysterol-binding protein could be related to the inhibitory effect of 25-hydroxycholesterol on DNA synthesis, which becomes critical when this sterol is added in the G2M phase of the cell cycle.     

p21Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes

Cyclin-dependent kinase inhibitor p21^Waf1 is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21^Waf1 in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21^Waf1-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21^Waf1 “tumor suppressor” having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21^Waf1 can be indispensable for complete oncogenic transformation.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.     

Synthesis of the nuclear protein cyclin does not correlate directly with transformation in quail embryo fibroblasts

The synthesis of the nuclear protein 'cyclin' (MW 36 000) has been analysed in several virus-transformed quail embryo fibroblasts (QEF) by means of quantitative two-dimensional gel electrophoresis. Our studies revealed that the synthesis of cyclin does not correlate directly with cell transformation, but with cell proliferation.     

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.     

Diethylstilbestrol induces arrest of rat vascular smooth muscle cell cycle progression through downregulation of cyclin D1 and cyclin E

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.