Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment.

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.

Tetracycline-controlled gene expression system achieves high-level and quantitative control of gene expression.

The tetracycline-controlled gene expression system utilizes the control elements of the tetracycline resistance operon encoded in TnlO of Escherichia coli to control gene expression in eukaryotic cells. Here we demonstrate the quantitative control of the expression of the luciferase gene, dihydrofolate reductase gene, and bcl-2 gene in HeLa S3 or Chinese hamster ovary AA8 cells using the tetracycline-controlled gene expression system. Regardless of the host cell lines or the genes being expressed, there is a common range of tetracycline concentration within which the expression of genes is most sensitively regulated. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system is higher than that of the wild-type CMV promoter/enhancer-driven system. Nonetheless, careful selection of stably transfected clones is necessary to achieve the optimally regulated gene expression using this system.

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

BCL-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells.

Apoptosis is a major form of cell death induced by chemotherapeutic drugs. Overexpression of the proto-oncogene bcl-2 can prevent apoptosis in various types of cells. We have constructed a HeLa S3 cell line in which the expression of bcl-2 can be controlled by the concentration of tetracycline in the medium. Using this system, we show that apoptosis induced by various cytostatic treatments could be delayed by the overexpression of bcl-2, as assayed by vital dye exclusion, apoptotic nuclei morphology, DNA histogram shift, and DNA fragmentation. Quantitative analysis revealed a hyperbolic curve when protection from apoptosis was plotted against the amount of Bcl-2. When cells were treated with aphidicolin for 12, 24, or 36 h and then replated in fresh media to assay for colony formation, the majority of cells that did not show apoptotic morphology at the time of drug removal failed to form colonies. Furthermore, Bcl-2 did not increase colony formation after 12-36 h of aphidicolin treatment. Therefore, with aphidicolin treatment, cells were committed to the death program upstream of the point of Bcl-2 action.

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.

Human cDNA clones that modify radiomimetic sensitivity of ataxia-telangiectasia (group A) cells.

Genes responsible for genetic diseases with increased sensitivity to DNA-damaging agents can be identified using complementation cloning. This strategy is based on in vitro complementation of the cellular sensitivity by gene transfer. Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disorder involving cellular sensitivity to ionizing radiation and radiomimetic drugs. A-T is genetically heterogeneous, with four complementation groups. We attempted to identify cDNA clones that modify the radiomimetic sensitivity of A-T cells assigned to complementation group [A-T(A)]. The cells were transfected with human cDNA libraries cloned in episomal vectors, and various protocols of radiomimetic selection were applied. Thirteen cDNAs rescued from survivor cells were found to confer various degrees of radiomimetic resistance to A-T(A) cells upon repeated introduction, and one of them also partially influenced another feature of the A-T phenotype, radioresistant DNA synthesis. None of the clones mapped to the A-T locus on chromosome 11q22-23. Nine of the clones were derived from known genes, some of which are involved in cellular stress responses. We concluded that a number of different genes, not necessarily associated with A-T, can influence the response of A-T cells to radiomimetic drugs, and hence the complementation cloning approach may be less applicable to A-T than to other diseases involving abnormal processing of DNA damage.

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.

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.

Life, death and genomic change in perturbed cell cycles.

HeLaS3 cells undergo apoptosis after 18-24 h of cell cycle stasis irrespective of the agent employed (colcemid, aphidicolin, cis-platin). At high drug concentrations apoptosis occurs in cells arrested in the cell cycle in which the drug is applied and at a cell cycle position dependent on the mechanism of drug action. At low concentrations (or short exposure times) cells undergo apoptosis after progressing through an aberrant mitosis and only after 18 h of cell cycle stasis in a 'pseudo G1/S' cell cycle position. Aberrent mitoses result in multipolar mitoses, chromosomal breakage and interchromosomal concatenation events. We propose that the ability of cells to delay progression into aberrent mitosis, as well as their propensity to undergo apoptosis, are important determinants of clinical cytotoxicity. We also suggest that apoptosis plays an important role in preventing the generation of aneuploidy and recombination and rearrangement events commonly associated with cancer.

Cyclin B1 expression in HeLa S3 cells studied by flow cytometry.

Using a procedure to stain cells simultaneously for cyclin B1 protein and DNA, we have examined cyclin B1 expression by flow cytometry in human cells under a variety of perturbing and nonperturbing conditions. The method described is useful for measuring relative differences in cyclin B level (immunochemically detectable epitope) as a function of cell cycle position on an individual cell basis and thus to examine cell cycle-related changes in cyclin B expression without prior cell synchronization. We show that in HeLaS3 cells, cyclin B1 accumulates in cells only after they become 4C and have resided in G2 for a short period of time. During colcemid-induced mitotic arrest cyclin B1 continues to accumulate in HeLa S3 cells, and under specific conditions of aphidicolin-induced unbalanced cell growth induced, cyclin B accumulates to supranormal levels prior to mitosis. Flow cytometric analysis of cyclin B expression and DNA content permits detailed examination of the effects of cell cycle perturbations on cyclin B expression under a variety of conditions.

The propensity for gene amplification: a comparison of protocols, cell lines, and selection agents.

We have studied cell lines of rodent and human origin for their propensity to become resistant to antifolates (methotrexate, trimetrexate), phosphonacetyl-L-aspartate (PALA), and colcemid, resistances associated with amplification of the DHFR, CAD, and MDR1 genes, respectively. We have employed two different methods: (1) a shallow step-wise selection protocol, where time to attain specified resistance is the quantitative measure, (2) the frequency of resistant colonies at specified drug concentrations. Although there are advantages and disadvantages to both methods, the two methods gave the same relative ranking of cell lines. Striking differences in the propensity for gene amplification (resistance) were found: human cell lines were less prone to amplify genes than Chinese hamster ovary (CHO) cells. This ranking was similar with all of the agents employed. Additionally, we observed that whereas PALA resistance in CHO cells is associated with amplification of the CAD gene, PALA resistance in the two human cell lines studied (HeLaS3 and VA13) was not associated with amplification and/or overexpression of the CAD gene, and thus this resistance to PALA occurs by an unknown mechanism.

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light.

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured as a function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction (approximately 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure to UV light, measured by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.

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.

In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal.

The cytotoxic neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) inhibits cell-cycle progression in CHO cells, affecting the G1/S and metaphase-anaphase transition points, as well as S phase. Mitotic arrest induced by ALLN is associated with the inhibition of cyclin B degradation. At mitosis-inhibiting concentrations of ALLN, cells undergo nuclear-envelope breakdown, spindle formation, chromosome condensation, and congression to the metaphase plate. However, normal anaphase events do not occur, and cells arrest in a metaphase configuration for a prolonged period. Steady-state levels of cyclin B increase to greater than normal mitotic levels, and cyclin B is not degraded for an extended period. Histone H1 kinase activity remains elevated during mitotic arrest. Duration of mitotic arrest depends on ALLN concentration; high concentrations (> 50 micrograms/ml) produce a prolonged mitotic arrest, whereas at lower concentrations, cells are transiently delayed through mitosis (up to 4-12 hr), after which they undergo aberrant cell division resulting in randomly sized daughter cells containing variable amounts of DNA. Cyclin B degradation fails to occur, and histone H1 kinase remains activated for the duration of mitotic arrest at all ALLN concentrations.

Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase.

Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases. Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s). However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734). To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein. These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity. Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily. The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones. Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.