Growth Arrest Conditions Research Articles

Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody.

The influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 microgram/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines.

Gas2Is a Multifunctional Gene Involved in the Regulation of Apoptosis and Chondrogenesis in the Developing Mouse Limb

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneideret al.,Cell 54, 787–793, 1988). In this study, the expression patterns of thegas2gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14.5-day mouse embryos. It was found thatgas2was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed thatgas2might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis,gas2is involved in the execution of the apoptotic program in hindlimb interdigital tissues—by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.

To be or not to be: the ultimate decision of the growth-arrested bacterial cell

The evolutionary survival of prokaryotes has depended not only on their performance during conditions that allow rapid growth, but also on their ability to adjust their internal affairs to enhance survival during prolonged periods of growth arrest. The small size of prokaryotes limits their ability to control their environment, and instead, their ability to cope with environmental challenges must rely on rapid and efficient control of gene expression. The majority of genes responding to a specific environmental condition causing growth arrest are, in general, uniquely induced by one specific stimulus. However, many genes, operons and regulons do not exclusively respond to one particular stress condition, and some regulons appear to be related in the sense that they share member genes. Furthermore, growth-arrest conditions activate several seemingly unlinked regulatory networks and several genes/proteins seem to respond to growth arrest in general. These proteins are interesting because we know very little about why growth-arrested, stationary phase cells ultimately die, and functional analysis of such general responders to growth arrest may help us to understand the genetic/biochemical basis for the bacterial defence against ageing processes. In this paper I review recent findings suggesting that several general responders to stasis form an integral part of a defence system aimed at avoiding the damaging effects of endogenously generated oxygen radicals. In addition, the curious finding that some proteins of a regulon are involved in enhancing the life-span of the growth-arrested cell while other members of the same regulon are devoted to killing it will be discussed.

To be or not to be: the ultimate decision of the growth-arrested bacterial cell

The evolutionary survival of prokaryotes has depended not only on their performance during conditions that allow rapid growth, but also on their ability to adjust their internal affairs to enhance survival during prolonged periods of growth arrest. The small size of prokaryotes limits their ability to control their environment, and instead, their ability to cope with environmental challenges must rely on rapid and efficient control of gene expression. The majority of genes responding to a specific environmental condition causing growth arrest are, in general, uniquely induced by one specific stimulus. However, many genes, operons and regulons do not exclusively respond to one particular stress condition, and some regulons appear to be related in the sense that they share member genes. Furthermore, growth-arrest conditions activate several seemingly unlinked regulatory networks and several genes/proteins seem to respond to growth arrest in general. These proteins are interesting because we know very little about why growth-arrested, stationary phase cells ultimately die, and functional analysis of such general responders to growth arrest may help us to understand the genetic/biochemical basis for the bacterial defence against ageing processes. In this paper I review recent findings suggesting that several general responders to stasis form an integral part of a defence system aimed at avoiding the damaging effects of endogenously generated oxygen radicals. In addition, the curious finding that some proteins of a regulon are involved in enhancing the life-span of the growth-arrested cell while other members of the same regulon are devoted to killing it will be discussed.

Trans-regulation of myogenin promoter/enhancer activity by c-ski during skeletal-muscle differentiation: the C-terminus of the c-Ski protein is essential for transcriptional regulatory activity in myotubes.

c-ski gene product is a nuclear protein with myogenesis-promoting and transforming activities. We have analysed the effects of c-ski transfection on the promoter/enhancer activity of the upstream region of the myogenin gene during in vitro myogenesis using CAT reporter assay. When co-transfected with c-ski into myogenic C2C12 cells, promoter/enhancer activity was efficiently suppressed in proliferating cells, but the myogenesis-induced increase in activity was potentiated approximately ten times more (150-fold in the ski-transfected cells) than the ordinary increase (12-fold in the mock) 48 h after induction of differentiation. In non-myogenic 10T1/2 cells, c-ski transfection caused persistent suppression of promoter/enhancer activity in both proliferating and growth-arrested (i.e. myogenesis-inducing) conditions. Thus the ski-dependent potentiation of myogenin gene transcriptional activity appears to be specific for myogenesis. The C-terminal region (amino acids 595-663) of the c-Ski protein was essential for the potentiating activity in myotubes. Other members of the ski-gene family, snoN and snoA, were ineffective in transactivation, possibly because of the defect in the corresponding C-terminal region. c-Ski protein underwent a mobility shift on SDS/PAGE after in vitro myogenesis which may explain the conversion of the activity from suppressive in myoblasts to potentiating in myotubes. Deletion analysis of the upstream region of the myogenin gene revealed that a responsive element to c-ski in myotubes is located at a distinct site upstream of the basal promoter/enhancer region.

Isolation of a novel cDNA whose corresponding mRNA is accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells

Four cDNA fragments, whose corresponding mRNAs were accumulated in growth-arrested confluent but not in growing sub-confluent rat 3Y1 cells, were isolated by the mRNA differential display method. Sequencing of the four cDNA fragments indicated that one of them was highly homologous to 3′ untranslated DNA region of mouse growth-arrest specific1 cDNA, while the other three (named c onfluent 3Y1 c ell- a ssociated No. 1 (cca1), cca2 and cca3) were novel. Of the three novel cDNAs, we presented some characteristics of cca2 cDNA: the cDNA consisted of 1795 nucleotides with a deduced open reading frame (ORF) encoding a protein of 338 amino acids, which showed a 35% identity with rat type IV 3 β-hydroxysteroid dehydrogenase. The CCA2 protein, with a molecular weight of 38 kDa, was accumulated in 3Y1 cells under growth-arrest conditions.

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair.

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.

Change in the expression of a nuclear matrix-associated protein is correlated with cellular transformation.

We have identified a monoclonal antibody that recognizes a nuclear matrix-associated protein in NIH 3T3 cells. The immunofluorescence pattern consists predominantly of bright nuclear granule clusters distributed throughout the nucleoplasm, with the exclusion of nucleoli. It recognizes a protein of 190 kDa that is down-regulated to various degrees in a panel of single-oncogene-transformed NIH 3T3 cells. Its localization is similar, but not identical, to the spliceosomal speckles. p190 shows a coordinate expression during the growth cycle of nontransformed NIH 3T3 cells; it is synthesized at the highest level under growth arrest conditions. It is expressed in adult mouse brain and is also present in human IMR-90 fibroblasts.

The Effects of Adverse Growth Conditions on the Shedding of Carcinoembryonic Antigen from Cultured LS180 Colon Cancer Cells

The synthesis and release of the tumor marker carcinoembryonic antigen (CEA) from the colon cancer cell line LS180 has previously been reported to be enhanced during the later stages of in vitro culture after growth has stopped. It has been suggested that CEA expression was inversely related to the growth rate for these cells (Kahan, B.D.; Rutzky, L.P.; Legrue, S.J.; Tom, B.H. Methods Cancer Res. 18:197-275; 1979 and Shi, Z.R.; Tsao, D.; Kim, Y.S. Cancer Res. 43:4045-4049; 1983). Our studies indicate, however, that while certain environmental perturbations that halt growth (e.g., glucose starvation and elevated temperatures) do indeed stimulate CEA expression and release; other growth-arresting conditions, such as oxygen starvation, have no effect. Replacement of spent or conditioned medium with fresh medium during the later culture stages resulted in a 10-fold decrease in CEA release, indicating that either depleted nutrients or accumulating cellular products (such as lactate or ammonium) trigger enhanced CEA production.

Mammalian Genes Coordinately Regulated by Growth Arrest Signals and DNA-Damaging Agents

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Hormonal and local factors influence antioxidant enzyme activity of rat fetal lung cells in vitro.

Monolayer cultures of fetal rat mixed lung cells respond to sublethal concentrations (50%) of oxygen by a reduced growth rate. Exposure to 95% O2 causes growth arrest and cell loss. In the presence of serum the addition of dexamethasone (5.5 nM), tri-iodothyronine (5.5 nM), or insulin (5 microU/ml) appeared to increase the cytotoxicity of 95% O2. Under growth-arrested conditions, in the absence of serum or elevated O2 concentrations, all three agents influence cellular antioxidant enzyme activities. Dexamethasone (0.055 nM) increased CuZn superoxide dismutase activity by 72% and glutathione peroxidase activity by 94%. Triiodothyronine (5.5 nM) increased CuZn superoxide dismutase activity 93%. Insulin (5 microU/ml) increased CuZn superoxide dismutase activity 90%, and catalase activity 58%. Dexamethasone, but not tri-iodothyronine or insulin, seems to have a protective effect against subsequent acute hyperoxia under serum-free conditions. Local non-hormonal factors may also influence lung cell responses to acute increases in oxygen concentrations, since cells acutely exposed to 50% or 95% O2 release a transferable factor(s) into their culture medium which increases antioxidant enzyme activities of non-hyperoxic lung cells.