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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 (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.

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.

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.

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.

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 G l/S’ cell cycle position. Aberrent mitoses result in miltipolar 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.

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 HeLa S3 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.

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.

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.

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

Epstein-Barr virus (EBV)-based vectors stably transform primate cells, which are expressing the trans-acting EBNA-1 gene, with efficiencies similar to those seen previously only in transient expression systems. These vectors propagate episomally and are readily recoverable into Escherichia coli. Taken together, these features make EBV vectors ideal for expression cloning. An EBV-based vector employing several advanced features that aid in the construction of highly representative, directionally cloned cDNA libraries is described. In a test of the system, an average of 45 colonies per 106 HPRT−, EBNA-1+ 293 cells per dish were generated following transfection with a HeLa cell cDNA library and selection for HPRT expression. This represents an increase in efficiency of 2 to 3 orders of magnitude over the efficiency of previously described systems using integrating vectors or EBV-based vectors in EBNA− cells.

We describe an Epstein-Barr virus (EBV)-based expression vector system for human cells that results in high transfection efficiencies (1–30%) in permissive human cell lines. The vector can replicate episomally for up to 6 months in a nonrearranged form. Human fibroblast cell lines are first rendered permissive by stable integration of an Epstein-Barr nuclear antigen-1 expression vector (CMV-EBNA). The EBV vector is a modification of that of B. Sugden et al. (1985, Mol. Cell. Biol. 5 , 410) and its functional elements, when introduced into “permissive” cells, include the EBV origin of plasmid replication, a drug resistance gene for selection in human cells (hygromycin), and bacterial sequences for maintenance in Escherichia coli . We have introduced an expression cassette for the production of high levels of mRNA from the RSV LTR such that transcription is in the same direction as replication fork progression. The high transfection frequency and maintenance in an episomal form allow for efficient transfection of complete cDNA libraries and for ready recovery of DNA sequences encoding selectable phenotypes.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.

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.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.

Methotrexate (MTX) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase (DHFR). MTX treatment of cultured cell lines leads to the emergence of resistant cell populations. Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of DHFR can be caused by DHFR gene amplification. We examined the effect of MTX on cells whose origin more closely approximates the in vivo condition by developing a culture system using dissociated brain tissue from 17-19 day old mouse embryos. At the first passage, cultures were divided into control and MTX groups. Cells were treated with the same or successively higher concentrations of MTX at each passage over a 3-4 month period. The first passage eliminated neurons and left a glial culture comprised of approximately 90% astrocytes. We used the Fluorescence Activated Cell Sorter in conjunction with fluorescent dyes to measure DHFR content, DNA content, size, and viability of glial cells following MTX treatment. MTX-treated cells divided but grew more slowly and were larger than untreated cells. Stepwise selection in 30/60/90 nM or 60/120 nM MTX resulted in significant two- to threefold increases in fluorescence, and hence DHFR levels. Slot hybridizations assays demonstrated a threefold increase in DHFR gene copy number in the DNA from the 30/60/90 cultures. Thus, our findings were consistent with the results obtained from somatic cell lines, and lend support to the hypothesis that gene amplification may be a common mechanism for the acquisition of resistance in many types of cells. They also indicate that glial cells may be a specific target for cytotoxic effects of MTX on the central nervous system.

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.

Publisher Summary Using flow cytometry to analyze asynchronous and synchronized cell populations, this chapter describes a number of features of drug-induced apoptosis in cultured cells: (1) apoptosis can occur in any cell cycle phase; (2) a given agent can induce apoptosis in more than one cell cycle phase; (3) there is a clear temporal relationship between cell cycle “arrest” induced by different agents and the onset of apoptosis; (4) proteolysis of specific intracellular proteins represents an early event of apoptosis; ( 5 ) aberrant mitosis precedes apoptosis under a variety of conditions, including exposure to inhibitors of DNA synthesis; (6) cells with sub-G, DNA content are not always apoptotic cells; (7) DNA content can be an unreliable index of cell cycle position; and (8) under all conditions, cell cycle “stasis” precedes drug-induced apoptosis. The chapter describes specific examples of applications of standard flow cytometric methods to the analysis of drug-induced apoptosis in HeLa S3 cells.

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 in 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.

For a number of years we have been examining the genotoxic and cytotoxic effects of antiproliferative drugs as well as cytotoxic treatments such as u.v. irradiation and hypoxia in cultured mammalian cells. The approach we have taken is to examine cellular response to treatment rather than drug-target interaction, and in particular, we have been interested in determining the relationship of treatment-induced cell cycle perturbation to cytotoxicity and genotoxicity (1–5).

The events of apoptosis represent the outcome of processes defining a common physiological pathway of dying cells, and the inputs to this pathway are complex and highly varied1–5. It is clear that the actions of cytotoxic agents in mammalian cells frequently feed into the apoptosis pathway, and an extremely wide variety of agents/treatments have been shown to induce the events of apoptosis in cultured mammalian cells6,7. It is important to note, however, that cells vary widely in responses to specific cytotoxic agents and the induction of apoptosis by a given agent can vary between cell lines.

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.

The identification of the genes responsible for the chromosomal breakage syndromes by their function has been discussed since the discovery of the common feature of these diseases, cellular sensitivity to specific DNA damaging agents. The advent of gene transfer methods supplied the experimental system for this approach, which is apparently simple and straightforward: Exogenous DNA is introduced in vitro into the patient’s cells, and selection is applied to identify cell clones showing increased resistance to the lethal action of the DNA damaging agent. An attempt is then made to rescue the piece of DNA responsible for this phenotypic change, which is expected to represent a candidate gene.

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Abnormalities in DNA synthesis and cell proliferation are characteristic of aged populations of proliferating cells. Cytogenetic analysis of jejunal crypt cells from young and senescent rats indicates that the imbalance in cell production in vivo is associated with an age-dependent increase in metaphase chromosome aberrations. Furthermore, the frequency of these chromosome aberrations is correlated with histologic evidence of cell death. These results demonstrate that the maintenance of genomic integrity is disturbed in the aged small intestine and may explain the age-related increase in the proportion of relatively undifferentiated villus epithelial cells in the small intestine of senescent rats.

We have used a microtiter assay to study the toxicity of various folate analogs in a series of cultured human cell lines that exhibit different degrees of resistance to methotrexate, an inhibitor of dihydrofolate reductase. These cells retain their sensitivity to the lipophilic antifolate BW301U despite the amplification of dihydrofolate reductase genes. Because the cell lines under investigation grow very slowly and have poor plating efficiencies in unconditioned medium, an assay was developed that relies on cell proliferation rather than colony formation as a measure of toxicity. This approach is easily generalized to provide a rapid and inexpensive assay of drug competition. Two-dimensional studies indicate that methotrexate and BW301U show differences in patterns of toxicity, competition, and rescue by folinic acid, suggesting that the two drugs act on different targets. Further applications of the microtiter assay to the analysis of multidrug interactions are discussed.

Using multiparameter flow cytometric analysis, we find that senescent cells accumulate in a unique cell-cycle compartment characterized in cell-cycle arrest in G1 and a significantly reduced nucleocytoplasmic ratio (genome size/cell mass) relative to cycling cells. With respect to gross cellular phenotype, the quiescent state of senescent cells differs from quiescence induced by density inhibition; the former is associated with a reduction in the nucleocytoplasmic ratio, while the latter is associated with an increase in the nucleocytoplasmic ratio. Senescent cells were present at all passages examined. The frequency of senescent cells was low in early-passage cultures and increased with passage number. Senescence of populations of IMR-90 cells reflects change in the relative frequency of these cells. The frequency of cells with karyotypic changes increased with the progressive accumulation of out-of-cycle cells.

A major obstacle to successful cytogenetic analysis of small intestinal crypt cells is the acquisition of a sufficient number of high-quality metaphases. A squash procedure has been developed for analysis of metaphase chromosomes from rat small intestine that largely circumvents this difficulty. The method involves a schedule of hypotonic treatment, fixation in ethanol: acetic acid, followed by maceration of the intestinal tissue in 3.5 N HCl. The procedure resulted in large numbers of well-spread, cytoplasm-free metaphases.

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

A complementary DNA synthesized from ovalbumin mRNA was used in hybridization experiments to study the early effect of estrogen and progesterone on the accumulation of ovalbumin mRNA sequences in the chick oviduct. Chicks treated with estrogen withdrawn from the hormone maintain a steady level of 60 molecules of ovalbumin mRNA per tubular gland cell, at least 80% of which are localized in the cytoplasm. After estrogen administration, there is a 3- to 4-hour lag before a rapid increase in the number of ovalbumin mRNA sequences and a parallel increase in ovalbumin synthesis. Progesterone causes a more rapid increase in both ovalbumin mRNA sequences and ovalbumin synthesis with a lag period of only 90 min. The hybridization results demonstrate that both estrogen and pregesterone affect the amount of ovalbumin mRNA per cell. The 3-hour lag period seen with estrogen appears to be caused by some event after the binding of the estrogen receptor to chromatin but prior to change in the rate of transcription of the ovalbumin gene.

The methotrexate-resistant AT-3000 line of S-180 cells has at least 150-fold more immunologically cross-reactive folate reductase than sensitive cells. Highly specific immunologic and protein purification procedures were used to show that the increased enzyme levels in this line are due to a corresponding increase in the rate of folate reductase synthesis. This observation indicates that the relative turnover of the enzyme is not significantly different in the two lines. Folate reductase was purified to homogeneity from both the sensitive and the methotrexate-resistant cells. Comparison of various physical, kinetic, and immunochemical properties of the enzymes revealed no differences. These observations suggest that the AT-3000 line contains one or more regulatory variations leading to the over-production of folate reductase protein that is similar, if not identical, to that produced by sensitive cells. In resistant cells, specific immunoprecipitation experiments demonstrated that folate reductase comprises as much as 7 to 8% of the continuously labeled soluble protein and 6 to 7% of the soluble protein synthesis. Growth of these lines in the absence of methotrexate resulted in a slow decrease in the level of folate reductase to less than 1%. This decrease corresponded to a similar decrease in the relative rate of enzyme synthesis. Variations in the level of folate reductase with cell growth are also due to changes in the relative rate of enzyme synthesis. In the AT-3000 line, pulse decay experiments showed that the half-life of folate reductase was long (50 hours) relative to cell doubling time (24 hours), and also that methotrexate had little or no effect on the turnover of the enzyme. Comparison of the incorporation of radioactive leucine into folate reductase in continuous and pulse labeling experiments gave independent confirmation of these results. Therefore, the relative rate of folate reductase synthesis was the major parameter determining the amount of folate reductase under all examined conditions that resulted in altered levels of the enzyme in resistant cells.

Pretreatment of 3T6 murine cells with the carcinogen UV radiation or N-acetoxy-N-acetylaminofluorene increased the number of methotrexate-resistant colonies. This carcinogen-induced enhancement was seen only at low toxicities. The enhancement was transient and was observed at its maximum when cells were subjected to methotrexate selection 12 to 24 h after treatment. The addition of a tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, during or after carcinogen treatment further enhanced this effect. A large proportion of the resistant colonies had an increase in the dihydrofolate reductase gene copy number and the relative proportions of colonies with amplified genes were similar, regardless of whether selected cells were untreated, treated with carcinogen, or treated with carcinogen plus promoter. We discuss some of the variables which both enhance the generation and improve the detection of methotrexate-resistant colonies, as well as certain implications of our results for the generation and mechanism of gene amplification.

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.

Exposure of Chinese hamster cells to reduced oxygen partial pressure results in a marked enhancement in the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. The frequency of enhanced resistance is a function of the length of exposure to hypoxic conditions and the time after recovery from hypoxia when cells are plated into methotrexate-containing medium. Hypoxia results in an inhibition of DNA synthesis; upon return to normal oxygen atmosphere, greater than 60% of cells in S phase at the time hypoxia was started subsequently undergo overreplication of DNA within a single cell cycle. The cells with the increased frequency of gene amplification are derived from this subset of overreplicated cells. These results are discussed within the context of the hypoxic state of many solid tumors and the high frequency of aneuploidy, chromosomal aberrations, and spontaneously occurring resistances to a number of cancer chemotherapeutic agents.

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.

We describe a bioassay for the quantitation of plasma methotrexate and trimetrexate levels employing intact cells. This assay is based on the intracellular saturation of dihydrofolate reductase with fluorescein-methotrexate (F-MTX) and its dose-dependent displacement by methotrexate or trimetrexate as monitored by flow cytometry. Serially diluted methotrexate-containing plasma, representing a wide chemotherapeutic range, produces F-MTX displacement curves similar to those of standard methotrexate solutions. There is no interference by normal plasma components such as folate and its reduced forms. Plasma methotrexate or trimetrexate concentration is the product of the 50% displacing concentration of standard antifolate (IC50) and the reciprocal of the plasma dilution which yields the same displacement. F-MTX competition with standard methotrexate displayed linear displacement from 18.0 ± 3.1 to 33.7 ± 1.5 n m (n = 10). The standard trimetrexate calibration curve was linear from 0.28 ± 0.03 to 1.5 ± 0.33 n m (n = 8). Thus, the bioassay sensitivities for methotrexate and trimetrexate are at least 18 and 0.3 n m , respectively. Comparison of methotrexate levels in 10 plasma specimens from cancer patients determined by the clinical enzyme inhibition assay and by our bioassay showed a high degree of correlation (r = 0.987).

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.

We have studied a line of 3T6 mouse embryo fibroblasts grown in progressively increasing concentrations of methotrexate. Resistance of cells to low concentrations of the inhibitor (less than 5 X 10(-5) M) is attributed to selective multiplication of the genes coding for dihydrofolate reductase and the resulting elevation of enzyme content. Cells isolated at a higher methotrexate concentration (4 X 10(-4) M) contain high levels of a dihydrofolate reductase with a reduced affinity for methotrexate. The altered dihydrofolate reductase exhibits a 270-fold reduction in binding affinity for methotrexate as measured by equilibrium dialysis (Kd = 5.4 X 10(-8) M versus 2 X 10(-10) M for the wild type enzyme). While binding to NADPH is unchanged, the Km for dihydrofolate is increased 3-fold over wild type enzyme and the turnover number for the reduction of dihydrofolate to tetrahydrofolate is decreased 20-fold. The altered dihydrofolate reductase shows a broader and more predominant acidic peak in its pH profile for this reaction. The molecular weights of the altered and wild type enzymes are identical as determined by sodium dodecyl sulfate-gel electrophoresis, but 2-dimensional electrophoresis reveals a significant basic shift in the migration of the altered enzyme. Studies with various folic acid analogs suggest that modifications involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases.