Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to less than 10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

Interaction of hyperthermia and metabolic inhibitors on the induction of chromosome damage in Chinese hamster ovary cells.

We have examined the chromosomal effects of heating asynchronously growing Chinese hamster ovary (CHO K1) cells in the presence of actinomycin D or cycloheximide. Actinomycin D was found to strongly potentiate the chromosome damaging effects of heat shock, an effect correlated with a strong nonadditive reduction in cell survival. In contrast, cycloheximide treatment reduced heat shock induced chromosome damage and resulted in a significant nonadditive increase in cell survival following heat shock. The different effects of these two inhibitors on chromosomal damage and cell survival are correlated in part with their effects on the rate of DNA synthesis during heat shock. The results suggest that an important aspect of the interaction of heat and metabolic inhibitors involves changes in cell cycle phase distribution of and/or progression through the S phase of the cell cycle induced by drug treatment prior to and during heat shock. The data indicate that the protective effect of cycloheximide in heat shocked cells may involve altered cell cycle progression and/or phase distribution of cells during hyperthermia.

Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation.

Although agents which act in a cell cycle phase specific manner are commonly used in the clinic and in basic research, it is as yet unclear why these agents are cytotoxic. In this paper, we examine the cellular events associated with the cytotoxicity of aphidicolin and vincristine in CHO strain AA8 cells. Cell killing resulting from aphidicolin treatment was found to require a period of inhibition-free growth following removal of the drug and was associated with characteristic aberrant mitotic processes. The cytotoxic effects of aphidicolin could be antagonized by the concomitant inhibition of protein synthesis with cycloheximide in the period of DNA synthesis inhibition. Cell killing resulting from treatment with vincristine was associated with the aberrant segregation of nuclear material and the formation of multiple partial nuclei. Vincristine cytotoxicity was found to be antagonized by concomitant administration of cycloheximide or cytochalasin D. These data support a hypothesis that the cytotoxic effects of cell cycle phase specific agents do not derive directly from their biochemical actions per se. We propose that cell death results from processes that are evoked by dissociation of normally integrated cell cycle events, and that dissociation involves replicative/mitotic events in the case of aphidicolin and karyokinetic/nuclear reformation events in the case of vincristine.

Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated methotrexate and daunorubicin.

We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry. Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining with fluoresceinated methotrexate and daunorubicin correlates well with drug resistance and expression of dihydrofolate reductase protein and the "classic" MDR phenotype in these cell lines. We show that flow cytometric analysis makes it possible to rapidly assess the potentially complex drug resistance phenotype(s) of cells selected with hydrophilic and lipophilic antifolates.

Heterogeneity in the mitotic checkpoint control of BALB/3T3 cells and a correlation with gene amplification propensity.

Chinese hamster ovary (and many rodent cell lines) transiently delay mitosis and progress into a second cell cycle without undergoing cytokinesis when treated with Colcemid, whereas HeLaS3 (and most human cell lines) arrest permanently in mitosis. We have discussed these differences and their consequences for cell survival under cell cycle-perturbing conditions within the context of mitotic checkpoint control (Schimke et al., Cold Spring Harbor Symp. Quant. Biol., 56: 417-425, 1991). Here, we report studies with mouse BALB/3T3 cell populations which, by the criterion of response to Colcemid, constitute a heterogeneous population with respect to mitotic checkpoint control. Clonal and subclonal populations retain population heterogeneity but with a bias for enrichment of cell populations that respond as do HeLaS3 cells. We have analyzed clones for their propensity for gene amplification as assessed by a stepwise increment selection protocol in methotrexate and report that there are significant differences in amplification propensities that correlate with differences in mitotic checkpoint control properties.

UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells.

Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein-4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2-q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain.

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.

PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.

Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay.

Malononitrilamides (MNAs) are a new class of immunomodulatory drug highly effective in in vivo models of allo- and xenotransplantation. Knowledge of their effects on immune cells, however, is limited and has been derived solely from investigations using isolated mononuclear cells. This use of purified cells to investigate drug activity is not ideal, so we have combined the analytical power of flow cytometry with our mitogen-driven, whole blood lymphocyte activation and proliferation assays to investigate the in vitro mechanism of action of MNAs. We first show that MNAs (A77 1726, HMR1279, and HMR1715), as well as brequinar (BQR) and cyclosporine (CsA), effectively inhibit cell activation antigen expression and lymphocyte proliferation. We next show that the inhibitory effects of MNAs and BQR, but not CsA, are reversed by the addition of uridine to the culture. These results suggest that inhibition of pyrimidine biosynthesis may be a mechanism by which MNAs suppress both lymphocyte activation and proliferation since these effects were reversed when uridine nucleotide pools were replenished. This novel finding of suppression of activation antigen expression by MNAs in whole blood expands our understanding of the effects of this new class of drug.

Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast.

Cell growth and division require the doubling of cellular constituents followed by their equal distribution to the two daughter cells. Within a growing population, the ratio of mitochondrial to cellular volume is maintained, as is the number of mitochondrial genomes per cell. The mechanisms responsible for coordinating nuclear and mitochondrial DNA synthesis, and for balancing increases in cell and mitochondrial size are not well understood. In studies of the fission yeast Schizosaccharomyces pombe we quantified cellular and mitochondrial DNA content by both Southern blot analysis and flow cytometry of cells stained with a variety of DNA-binding fluorochromes, which we show are able to detect nuclear and mitochondrial DNA with different efficiencies. In the conditional cell division cycle mutant cdc10, which is unable to initiate nuclear DNA synthesis, we found that there was an increase in the mitochondrial DNA content in the absence of nuclear DNA replication. This demonstrates that mitochondrial and nuclear DNA synthesis are not obligately linked. We also show that mitochondrial DNA replication is not required for the increase in mitochondrial size that occurs as cells elongate, although this results in a decrease in the ratio of mitochondrial DNA to mitochondrial volume.

Telomerase, checkpoints and cancer.

Telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state. In particular, the highly specific correlations and early causal relationships between telomere loss in the absence of telomerase activity and replicative senescence or crisis, on the one hand, and telomerase reactivation and cell immortality, on the other, point to a new and important paradigm in the complementary fields of ageing and cancer. Although the signalling pathways between telomeres and transcriptional and cell cycle machinery remain undefined, recently described homologies between telomeric proteins and lipid/protein kinase activities important in chromosome stability provide evidence for the existence of pathways transducing signals originating in chromosome structure to cell cycle regulatory processes. Similarities between cell cycle arrest at senescence and the response of mortal cells to DNA/oxidative damage suggest overlap in the signal transduction mechanisms culminating in irreversible and stable cell cycle arrest. The feasibility of targeting telomeres/telomerase as a strategy for antiproliferative therapeutics has been shown in studies in yeast, in which mutations in specific telomere associated genes result in delayed cell death. Similarly, antisense oligonucleotide inhibition of telomerase activity in human tumour cells (HeLa) results in delayed cell death. The mechanism of cell death and possible escape from this fate require further study. In human cells, however, it would seem reasonable to predict that in these circumstances, apoptosis is induced in the vast majority of cells either directly in response to a DNA damage signal arising from critically shortened telomeres or as a secondary consequence of genetic instability.

Genome evolution in pocket gophers (genus Thomomys). I. Heterochromatin variation and speciation potential.

A basic dichotomy exists in the amount and chromosomal position of constitutive heterochromatin (C-bands) in species of pocket gophers, genus Thomomys. Members of the "talpoides-group" of species (e.g., T. talpoides and T. monticola) have C-bands restricted to the centromeric regions. These taxa are characterized by Robertsonian patterns of karyotypic evolution. In contrast, species within the "bottae-group" are characterized by extensive amounts of heterochromatin, placed as whole-arm and apparent whole-chromosome (T. bottae) or as large interstitial blocks (T. umbrinus). These species are characterized by extensive non-Robertsonian variation in karyotype, variation which may be expressed from local population polymorphism to between population or species polytypy. Within T. bottae, the number of whole-arm heterochromatic autosomes is inversely proportional to the number of uniarmed chromosomes in the complement, which ranges from 0 to 36 across the species populations. In all-biarmed karyotypic populations, upward to 60 percent of the linear length of the genome is composed of heterochromatin. Populations with extensive heterochromatin variation and those with similar amounts meet and hybridize freely in nature. The implications of these date for current ideas on the function of heterochromatin, particularly as related to speciation models, are discussed.

Genome evolution in pocket gophers (genus Thomomys). II. Variation in cellular DNA content.

Cellular DNA content (2 C-value) was measured by fluorescence flow cytometry of chromomycin-A3 stained spleen cells in 2 subgenera, 5 species, and 21 subspecies of pocket gophers (genus Thomomys). The data indicate that, in Thomomys: (1) interspecific variation is extensive but, while some congeneric species differ by as much as 230%, others are identical in C-value: (2) intraspecific differentiation can be extensive with C-values differing by as much as 35%; and (3) populations of the same subspecies with apparently similar karyotypes can differ significantly in C-value. The implications of these results for hypotheses of the "adaptive" significance of C-value variation and genome evolution are discussed.

Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization.

We have studied the effect of the cell synchronization agents compactin, ciclopirox olamine, mimosine, aphidicolin, ALLN, and colcemid on several parameters of cell cycle progression in mitotically synchronized HeLa S3 cells. Using cell size and cyclin A and B levels as markers of cytoplasmic progression and DNA content as a measure of nuclear cell cycle position, we have examined coordination of cytoplasmic and nuclear events during induction synchrony. Each synchronizing agent was unique in its effect on the coordination of the cytoplasmic and nuclear cycle. Mimosine, aphidicolin, ALLN, and colcemid disrupted cell cycle integration while compactin and ciclopirox olamine did not. Continued net cell growth during cell cycle arrest was the most dramatic in aphidicolin-treated cells, which averaged a 60% increase in size. Mimosine, ALLN, and colcemid produced an increase in cell size of approximately 25%, and ciclopyrox olamine and compactin exerted a negligible effect. Cyclin A and B were found at mitotic (high) or G1 (low) levels, or in combination of high and low concentrations not correlated with DNA content in drug-treated cells. For example, treatment with mimosine, which arrests cells in G1 with 2C DNA, resulted in cyclin A accumulating to mitotic levels, whereas cyclin B remained at a low concentration, the first time this phenomenon has been observed. These results demonstrate that populations of synchronized cells obtained by different drug treatments are blocked at biochemically distinct cell cycle points not apparent by cytometric measurement of DNA content. Our results provide conclusive evidence that induced synchrony methods differ with respect to their impact on cell cycle organization and from the pattern seen with nonperturbing cell selection methods.

Farnesyl acetate, a derivative of an isoprenoid of the mevalonate pathway, inhibits DNA replication in hamster and human cells.

Cells treated with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme which catalyzes the rate-limiting step of the mevalonate pathway, are arrested prior to the DNA synthesis (S) phase of the cell cycle. Identification of a specific pathway product or products with a role in DNA replication, however, has remained elusive. In this report we demonstrate that farnesyl acetate, a derivative of the key isoprenoid pathway intermediate farnesyl pyrophosphate, inhibits DNA replication in both Chinese hamster ovary cells and human (HeLa) cells. This effect is revealed by measurement of DNA content using fluorescence-activated cell sorter analysis and by measurement of [3H]thymidine incorporation. We show that cells treated with farnesyl acetate retain protein synthesis capacity as DNA replication is inhibited and remain intact as viewed with the vital stain propidium iodide. The inhibition of DNA replication by farnesyl acetate occurs in cells treated with high levels of compactin and in cells lacking HMG-CoA reductase. These results indicate that farnesyl acetate action is not dependent on metabolism through the isoprenoid pathway and is not the result of the loss of a metabolite required for replication nor the accumulation of a metabolite which is inhibitory. In addition, cells treated with farnesyl acetate for over 6 h are irreversibly blocked from progressing through S phase, a phenomenon which differs sharply from the results with compactin, removal of which results in synchronous progression through S phase. Farnesyl acetate also blocks protein prenylation in cells, to a degree comparable to a known farnesylation inhibitor, BZA-5B. We propose that farnesyl acetate is acting in a manner quite different from the metabolic block caused by compactin, causing a rapid and irreversible block of DNA replication.

Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.

This paper reports that there are major differences between mammalian cell lines in the propensity to progress into subsequent cell cycles when mitosis is inhibited with agents that disrupt the assembly of the mitotic spindle apparatus (Colcemid, nocodazole, and taxol). Human HeLa S3 cells, which represent one extreme, remain arrested in mitosis, with elevated levels of cyclin B and p34cdc2 kinase activity. In Chinese hamster ovary cells, at the other extreme, the periodic rise and fall of cyclin B levels and p34cdc2 kinase activity is only transiently inhibited in the absence of mitosis. The cells progress into subsequent cell cycles, without dividing, resulting in serial doublings of cellular DNA content. In general, the propensity to progress into subsequent cell cycles in the absence of mitosis appears to be species related, such that human cell lines remain permanently blocked in a mitotic state, whereas rodent cell lines are only transiently inhibited when spindle assembly is disrupted. We interpret these results to indicate that in mammalian cell lines there exists a checkpoint which serves to couple cell cycle progression to the completion of certain karyokinetic events. Furthermore, either such a checkpoint exists in some cell lines but not in others or the stringency of the control mechanism varies among different cell lines.

Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells.

We have studied the relationship between apoptosis and drug-induced cell cycle perturbation in HeLa S3 cells when treated with the anti-tubulin drug colcemid. We found that at least two distinct mechanisms contributed to colcemid cytotoxicity and apoptosis. Continuous exposure to concentrations of colcemid sufficient to block cells at the mitotic checkpoint led to the appearance of apoptotic cells approximately one cell cycle after their initial accumulation in mitosis. Continuous exposure to concentrations sufficient to delay mitotic progression but insufficient to cause mitotic arrest, or pulse exposure to concentrations of colcemid sufficient to induce mitotic block, led to the generation of multipolar mitoses and genetically deficient hypodiploid daughter cells which underwent apoptosis while in interphase. The fact that aberrant spindle function delayed but did not block cells at the mitotic checkpoint indicates that the mitotic checkpoint senses the presence or absence of the spindle but not spindle abnormalities. In both mitotic and interphase cells, colcemid-induced apoptosis occurred after a period of cell cycle stasis during which cells failed to complete an initiated cell cycle. These results are discussed with reference to understanding the relationship between apoptosis and the regulation of cell cycle progression.

Differences in the regulation of protein synthesis, cyclin B accumulation, and cellular growth in response to the inhibition of DNA synthesis in Chinese hamster ovary and HeLa S3 cells.

We have shown previously that there are significant differences between mammalian cell lines in response to disruption of the assembly of the mitotic spindle apparatus (Kung, A. L., Sherwood, S. W., and Schimke, R. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9553-9557). In this paper we report that there are also significant differences between mammalian cell lines in response to the inhibition of DNA synthesis. In HeLa S3 cells protein synthesis is down-regulated, and cellular growth is arrested in response to the inhibition of DNA synthesis. Upon release from inhibition and resumption of normal growth, cellular viability is maintained near untreated control levels. In contrast, Chinese hamster ovary cells continue to accumulate protein and continue to undergo cellular growth during the period of DNA synthesis inhibition. Cyclin B levels accumulate throughout the period of inhibition and rapidly exceed normal levels at mitosis. The degree of aberrant growth during the period of transient DNA synthesis inhibition is directly related to the degree of subsequent cytotoxicity. If protein accumulation and cellular growth are limited with partially inhibitory levels of cycloheximide during the period of DNA synthesis inhibition, the cytotoxic effects are abolished. These results support the concept that aberrant growth and accumulation of proteins during a transient period of DNA synthesis inhibition are primary determinants of subsequent cell killing.