Oncogenic c-H-ras deregulates survivin expression: an improvement for survival

Survivin protein accomplishes two basic functions: cell cycle regulation and control of apoptosis. It is only expressed in G2/M phase and it influences rescue pathways in apoptosis-induced cells. Overexpression of constitutive active c-H-ras in HeLa, or induction of c-H-ras in a stable HeLaDiR cell line, led to sustained survivin expression in all cell cycle phases and even protected cells from drug induced apoptosis. siRNA-mediated silencing of survivin reversed this protection. Here we link the anti-apoptotic property of survivin to its cell cycle (in)dependent regulation via the activity of oncogenic c-H-ras.     

Induction of survivin expression by taxol (paclitaxel) is an early event, which is independent of taxol-mediated G2/M arrest

Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G(2)/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5-15-fold) within 4 h without G(2)/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G(2)/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G(2)/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics.     

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.     

Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.

BACKGROUND: Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS: We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS: Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.     

Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle

Induction of c-fos mRNA levels associated with the stimulation of growth by fetal bovine serum following quiescence was examined in three cell types following brief (24 h) serum starvation. Starved NIH-3T3 and HeLa S3 cells experienced c-fos mRNA induction 20-30 min after addition of serum. In contrast, Swiss-3T3 cells expressed c-fos constitutively following serum starvation. The pattern of oncogene expression coincided with the level of quiescence of each cell line prior to induction. Serum inductions of c-fos expression was dependent upon the response of each cell line to serum starvation, c-fos expression was also examined in HeLa S3 cells that had been separated into sequential cell cycle phases by centrifugal elutriation, c-fos expression peaked during the earliest part of the synchronous G1 phase. The amount of c-fos mRNA measured was approximately twice that found during other cell cycle phases. This suggests that, in addition to its role during the transition from quiescence, the c-fos gene product may play a regulatory role during the earliest part of G1 phase of the continuous cell cycle.     

Regulation of survivin by retinoic acid and its role in paclitaxel-mediated cytotoxicity in MCF-7 breast cancer cells

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.     

Differential susceptibility of na?ve and activated human gammadelta T cells to activation-induced cell death by T-cell receptor cross-linking.

BACKGROUND: T cells undergo activation-induced cell death (AICD) through repeated stimulation of their T cell receptors (TCRs). Activated human gammadelta T cells were found to die by apoptosis when their TCRs were cross-linked by antibodies, whereas naïve gammadelta T cells freshly isolated from blood did not. Therefore, we investigated the factors that could contribute to this differential susceptibility. MATERIALS AND METHODS: Gammadelta T cells were isolated from the peripheral blood of healthy human volunteers and their TCRs were cross-linked either directly (naïve) or after an in vitro incubation of 11 days (activated). Their cell cycle profiles, cytokine, Fas and FasL mRNA messages, and surface expression of Fas and FasL were determined. RESULTS: The naïve cells were cycling while the activated T cells exited from the G1 to subG1 phase upon TCR cross-linking. IL-2 and IL-4 mRNAs and surface expression of FasL were detected only in activated T cells in the time period examined. In addition, cFLIP mRNA expression was found only in naïve gammadelta T cells and activated T cells treated with cyclosporin A (CsA), which inhibited AICD in the activated T cells. CsA also downregulated the surface expression of FasL in activated T cells. CONCLUSIONS: The differential expression of cytokines, apoptotic inducers and inhibitors provide the basis for the differential susceptibility of naïve and activated gammadelta T cells to AICD upon TCR cross-linking. This contributes to our understanding of the regulation and maintenance of gammadelta T-cell homeostasis, which would be important in many infectious as well as autoimmune diseases, where gammadelta T cells have been implicated.     

Mapping G1 phase by the structural morphology of the prematurely condensed chromosomes

The object of this study was to develop a map of G1 phase on the basis of the progressive changes taking place in the morphology of the prematurely condensed chromosomes as the cells traverse through G1 and then use this technique to determine the cell cycle location of normal and transformed cell populations in plateau phase. The morphology of the prematurely condensed chromosomes (PCC) of G1 cells in random populations was found to be highly variable. For a better understanding of the relationship between the morphology of the G1-PCC and their position within G1 phase, synchronized populations of Chinese hamster ovary (CHO) cells in early, mid-, and late G1 phase were fused with mitotic cells. Early G1 cells resulted in highly condensed G1-PCC, while late G1 cells gave very extended G1-PCC. Mid-G1 cells resulted in PCC of intermediate condensation. To test the validity of these criteria for mapping the position of a cell in the cell cycle, synchronous G1 cell populations were treated with a variety of metabolic inhibitors. Cycloheximide and actinomycin D were shown to block cell in early G1 phase, while excess thymidine and hydroxyurea blocked cells in early S phase. The results presented here indicate that, upon reaching plateau phase, normal cell populations (BALB-C mouse 3T3, human PA-2, and WI 38) stop in early G1, while most cells in transformed cell lines (CHO, HeLa, and mouse SV-3T3) accumulate in late G1.     

Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.     

An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7

Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.     

Ras is active throughout the cell cycle,but is able to induce cyclin D1 only during G2 phase

The control of cell cycle progression has been studied in asynchronous cultures using image analysis and time lapse techniques. This approach allows determination of the cycle phase and signaling properties of individual cells, and avoids the need for synchronization. In past studies this approach demonstrated that continuous cell cycle progression requires the induction of cyclin D1 levels by Ras, and that this induction takes place during G2 phase. These studies were designed to understand how Ras could induce cyclin D1 levels only during G2 phase. First, in studies with a Ras-specific promoter and cellular migration we find that endogenous Ras is active in all cell cycle phases of actively cycling NIH3T3 cells. This suggests that cyclin D1 induction during G2 phase is not the result of Ras activation specifically during this cell cycle period. To confirm this suggestion oncogenic Ras, which is expected to be active in all cell cycle phases, was microinjected into asynchronous cells. The injected protein induced cyclin D1 levels rapidly, but only in G2 phase cells. We conclude that in the continuously cycling cell the targets of Ras activity are controlled by cell cycle phase, and that this phenomenon is vital to cell cycle progression.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Human survivin is a kinetochore-associated passenger protein

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.     

The cell cycle dependence of thermotolerance. I. CHO cells heated at 42 degrees C

We examined the dependence of heat killing and thermotolerance on the position and progression of Chinese hamster ovary (CHO) cells in the cell cycle. We measured cell cycle perturbations and survival of asynchronous and synchronized G1-, S-, and G2-phase cells resulting from continuous heating at 42.0 degrees C for up to 80 hr. Thermotolerance under these conditions was transient in nature, was dependent on the position of cells in the cell cycle, and occurred concurrently with a heat-induced delay of progression of G1- and G2-phase cells. When G1 cells were heated, survival decreased to 25% after 4 hr, at which time the thermotolerance was expressed. For G2 cells survival decreased initially at the same rate (T0 congruent to 3 hr) but thermotolerance was not expressed until approximately 12 hr, at which time the survival was 4%. The rate of decrease in survival was much more rapid for cells heated in mid-S phase (T0 congruent to 0.5 hr), and these cells did not express thermotolerance at a measurable level. Concurrent with the expression of thermotolerance, the progression of cells heated in G1 and G2 was delayed. Following the expression of tolerance, progression resumed at a rate approximately equal to the rate of decrease in survival of the G1 population. Cells heated in mid-S phase continued to progress through the cell cycle until they reached G2, where they were also delayed.     

The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells

T cells can undergo activation-induced cell death (AICD) upon stimulation of the T cell receptor-CD3 complex. We found that the extracellular signal-regulated kinase (ERK) pathway is activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the ERK pathway inhibited FasL expression during AICD, whereas activation of the ERK pathway with a constitutively active MEK1 resulted in increased expression of FasL. We also found that pretreatment with the specific MEK1 inhibitor PD98059 prevented the induction of FasL expression during AICD and inhibited AICD. However, PD98059 had no effect on other apoptotic stimuli. We found only very weak ERK activity during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the orphan nuclear steroid receptor Nur77, which has been shown to be essential for AICD. In conclusion, this study demonstrates that the ERK pathway is required for AICD of T cells and appears to regulate the induction of Nur77 and FasL expression during AICD.