Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").     

Influence of carbon catabolite repression on the G1 arrest of Saccharomyces cerevisiae MATa cells by alpha factor

Cells of the yeast Saccharomyces cerevisiae of the a mating type were arrested at the G1 phase of the cell division cycle after treatment with alpha factor in a culture medium containing a high concentration (2%, w/v, or higher) of a catabolite-repressing sugar. In media containing either a lower concentration of sugar or a non-fermentable carbon source, the extent of G1 arrest induced by the pheromone was reduced or became undetectable. Under catabolite-derepressing conditions alpha factor was inactivated by a cells at a higher rate than that found in repressing media. These results indicate the existence of a close correlation between the action of alpha factor on a cells and conditions of catabolite repression or derepression. A joint mechanism of action of alpha factor and catabolite-repressing carbon sources on a cells is postulated.     

Growth factor-induced cell division is paralleled by translocation of Gi alpha to the nucleus

Induction of mitosis by certain growth factors is inhibited by pertussis toxin, indicating that the GTP-binding protein, Gi, is involved in receptor signal transduction to initiate cell division. However, the substrates of receptor-activated Gi that are involved in mitosis have not been determined. The present study has examined whether Gi may directly modulate cell division by receptor-induced subcellular translocation of the alpha subunit of Gi (Gi alpha). Insulin and EGF, particularly when added together or in combination with phorbol dibutyrate (PdBu), induced a rapid (1-4 h) redistribution of Gi alpha from the plasma membrane to perinuclear sites in the cell. After 2 days of stimulation, Gi alpha had translocated into the nucleus of dividing cells and bound specifically to the separating chromatin of dividing nuclei. Unstimulated cells did not display translocation of Gi alpha. This demonstrates a direct involvement of Gi alpha in cell division, which provides an apparently uninterrupted link from growth factor receptor to nucleus.     

Effect of alpha factor pheromone on the activity of enzymes which catalyze the synthesis and hydrolysis of cell wall structural polymers from Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae of the a mating type treated with alpha factor contain an increased amount of structural polymers (beta-glucans and chitin) in their cell walls and, consequently, exhibit higher glucan synthetase and chitin synthetase activities than untreated cells. However, alpha factor has no detectable effect on the activities of these enzymes when they are assayed, "in vitro", in the presence of the pheromone. On the other hand, the activity of beta-glucanases remains constant during the time that growth of a cells is kept arrested by alpha factor at the G1 phase of the cell division cycle and starts to increase when budding of the cells is reinitiated.     

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.     

Comparison of dose-response curves for alpha factor-induced cell division arrest, agglutination, and projection formation of yeast cells. Implication for the mechanism of alpha factor action

MAT alpha cells of the yeast Saccharomyces cerevisiae produce a polypeptide mating pheromone, alpha factor. MATa cells respond to the pheromone by undergoing several inducible responses: the arrest of cell division, the production of a cell surface agglutinin, and the formation of one or more projections on the cell surface commonly termed the "shmoo" morphology. Dose-response curves were determined for each of these inducible responses as a function of alpha factor concentration. It is shown that under conditions commonly employed in previous studies, the dose-response for cell division arrest is determined by the rate at which cells inactivate the alpha factor. In order to achieve conditions where inactivation would not be the dominant parameter, the cell division response to alpha factor was monitored at low cell densities. Under conditions of essentially no alpha factor destruction, the dose of alpha factor at which cells exhibit a half-maximal response for cell division arrest (2.5 X 10(-10) M) is nearly the same as that at which cells exhibit a half-maximal response for agglutination induction (1.0 X 10(-10) M). On the contrary, the half-maximal response for projection formation was obtained at doses of alpha factor 2 orders of magnitude higher (1.4 X 10(-8) M). These results are consistent with the same high affinity alpha factor receptor mediating both cell division arrest and agglutination induction. A different system of lower affinity must mediate projection formation. Alternatively, if the same system and receptor are used, then a much higher occupancy is required for the induction of projections compared to division arrest and agglutination induction.     

Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrested in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.     

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

Induction of the yeast alpha-specific STE3 gene by the peptide pheromone a-factor

The yeast Saccharomyces cerevisiae exhibits two mating types, a and alpha. Efficient mating of a and alpha cells requires the action of peptide pheromones secreted by each cell type. For example, a cells secrete a-factor, which alters the physiology of alpha cells, thereby preparing those cells for mating. To investigate the mechanism by which the pheromones act on the target cells, we have examined the effect of a-factor on expression of the STE3 gene, a gene which is required for mating by alpha cells and which is expressed only in alpha cells. We have monitored STE3 expression by two assays: RNA production from the chromosomal STE3 locus and beta-galactosidase activity produced from a plasmid-borne STE3-lacZ gene fusion. By both assays we show that a-factor induces a rapid increase in STE3 expression. Induction of STE3 RNA occurs even if protein synthesis is blocked by cycloheximide. Using temperature-sensitive cell division cycle mutants, we have also shown that induction occurs in cells arrested at several discrete positions in the cell cycle. These results demonstrate (1) that induction of STE3 expression by a-factor is a primary response to the pheromone, and (2) that alpha cells are capable of responding to a-factor regardless of their position in the cell cycle.     

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.     

Arrested states produced by isoleucine deprivation and their relationship to the low serum produced arrested state in Swiss 3T3 cells

This study examines cell cycle maturational arrests induced by isoleucine deficiency in Swiss 3T3 cells. Whereas low serum selectively blocks the maturation of cells in mid-G1, while allowing late G1, S, G2 and M cells to continue maturing through the cell cycle, isoleucine deprivation blocks the maturation of cells at the end of G1 and also in late S. These blocks are at median ages of approx. 5.2 ± 1.3 h and 12.1 ± 3 h after division. Cells prevented from maturing beyond these two points require serum to resume proliferation, although they have passed the mid-G1 low serum block point. This indicates that resumption of proliferation requires additional events not part of normal transit through the cell cycle. Furthermore, the kinetics of growth resumption differ from those of low serum arrested cells. Cells arrested by isoleucine deprivation appear to be in physiological states which are not part of the proliferative cell cycle and which do not coincide with the low serum arrested state.     

A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways.     

Rapid acquisition of an enhanced capacity to produce tumor necrosis factor, alpha/beta interferon, and interleukin 6 after implantation of tumor cells.

This study shows that the ability of mice to produce tumor necrosis factor (TNF), alpha/beta interferon (IFN-alpha/beta), and interleukin 6 (IL-6), but not interleukin 1 (IL-1), in response to endotoxin was dramatically augmented within 24 h of intradermal implantation of 10(6) tumor cells. Tumor cell implantation also caused endotoxin-independent appearance of IFN-alpha/beta and IL-6 in serum within 24 h. Priming for endotoxin-induced TNF production was not evident during the first 12 h of tumor cell implantation and it had decreased by 72 h. However, this decrease was followed by a second peak of priming on day 6 of tumor growth. Priming for endotoxin-induced TNF production was not induced by injection of dead tumor cells, the products of live tumor cells, or syngeneic or allogeneic splenocytes. Priming for TNF production was associated with an increased susceptibility of mice to endotoxin toxicity. These data suggest the existence of a cytokine-dependent host defense mechanism that is rapidly elicited in response to tumor cell implantation.     

Requirement for mevalonate in cycling cells: quantitative and temporal aspects

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.