RNA synthesis in Chinese hamster cells. II. Increase in rate of RNA synthesis during G1.

Cultures of mitotic Chinese hamster cells, prepared by mechanical selection, were pulse-labeled with methionine-methyl-(14)C or with uridine-(3)H at different stages in the life cycle. The rate of (14)C incorporation into 18S RNA was measured, as was the rate of uridine-(3)H incorporation into total RNA for both monolayer and suspension cultures. The rate of incorporation increased continuously throughout interphase in a fashion inconsistent with a gene-dosage effect upon RNA synthesis.

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G(1)-Arrested Cells in Suspension Culture.

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G(1) following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G(1), indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10(-6)M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10(5) cells/ml. Under similar conditions, about 4 x 10(-6)M isoleucine was required for all G(1)-arrested cells to progress through cell division. In contrast, 1 x 10(-4)M glutamine was necessary for maximum initiation of DNA synthesis in G(1) cells, along with sufficient isoleucine. A technique for rapid production of G(1)-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G(1) after 30 hr.

Regulation of initiation of DNA synthesis in Chinese hamster cells. I. Production of stable, reversible G1-arrested populations in suspension culture.

Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8-9 x 10(5) cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G(1) Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.

The effect of thymidine on the duration of G1 in Chinese hamster cells.

The generation time of a Chinese hamster cell line was varied by the use of different lots of sera in the culture media. Analysis of the division waves following thymidine synchronization showed that lengthening of the generation time was a result of an increase in duration of the G(1) phase and that thymidine treatment reduced the duration of G(1) back to its minimum value.

RNA synthesis in Chinese hamster cells. I. Differential synthetic rate for ribosomal RNA in early and late interphase.

The incorporation of methionine-methyl-(14)C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1-1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.

Cell cycle-specific changes in histone phosphorylation associated with cell proliferation and chromosome condensation.

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G(1)-arrested and early G(1)-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G(2)/M boundary and traversing prophase. Since these phosphorylation events do not occur in G(1), S, or G(2) and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled (32)PO(4) from those specific histone fractions during transition of metaphase cells into interphase G(1) cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.

A method for comparing effects of different synchronizing protocols on mammalian cell cycle traverse. The traverse perturbation index.

After treatment of Chinese hamster cells (line CHO) with various protocols for synchrony induction, the subsequent ability of cells to traverse the cell cycle (i e., to perform, an essential cell cycle process) has been determined by measurement of the DNA distribution pattern among cells in large populations with the Los Alamos flow microfluorometer In the cultures prepared by the various synchronizing techniques the vast majority of cells traversed the cell cycle in a normal fashion; however, in all cultures examined there remained small subpopulations which, though remaining viable for several days, could not carry out normal traverse. After reversible inhibition of DNA synthesis by means of a double-thymidine blockade, approximately 17% of the cells were unable to complete genome replication. After reversal of G(1) arrest resulting from cultivation of cells in isoleucine-deficient medium, 12 4% of the cells commenced synthesis of DNA but were unable to complete the S phase. Cells prepared by mitotic selection yielded a subpopulation (5 5% of the total cells) with a G(1) DNA content which remained viable but noncycling for at least 5 days. We propose a term "traverse perturbation index" which is defined as the fraction of cells converted to a noncycle-traversing state as the result of experimental manipulation. A knowledge of the perturbation index will allow direct comparison of effects on cell cycle traverse of various synchrony-induction protocols

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry.

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three-color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein-fluorescein isothiocyanate (green), and RNA-bound pyronin Y (red) fluorescence measurements were correlated as each stained cell intersected three spatially separated laser beams. The analytical scheme provided sensitive and accurate fluorescence determinations by minimizing the effects of overlap in the spectral characteristics of the three dyes. Computer analysis was used to generate two-parameter contour density profiles as well as to obtain numerical data for subpopulations delineated on the basis of cellular DNA content. Such determinations allowed for analysis of RNA to DNA and RNA to protein ratios for cells within particular regions of the cell cycle. The technique was used to study the interrelationship of DNA, RNA, and protein contents in exponentially growing Chinese hamster ovary cells as well as in cell populations progressing the cell cycle after release from arrest in G1 phase. The sensitivity of the method for early detection of conditions of unbalanced growth is demonstrated in the comparison of the differential effects of the cycle-perturbing agent, adriamycin, on cells treated either during exponential growth or while reversibly arrested in G1 phase.

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210.

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

Cell-cycle analysis in 20 minutes.

Mithramycin added to mammalian cells fixed in aqueous ethanol is bound to DNA and fluoresces in direct proportion to the cellular DNA content. Quantitative fluorescence measurement by means of a high-speed flow system instrument provides a rapid method for cell-cycle analysis and for the first time permits continuous monitoring of cell-cycle kinetics during ongoing experiments.

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry.

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

Inducibility of metallothionein throughout the cell cycle.

Synchronized Chinese hamster cells were induced with ZnCl2 at multiple stages of the cell cycle and labeled with [35S]cysteine, and the 35S-labeled proteins were isolated and separated into metallothionein and nonmetallothionein fractions. Metallothionein was found to be inducible in all stages of the cell cycle and in G1-arrested cells.

Cell-cycle-specific chromosome damage following treatment of cultured Chinese hamster cells with 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl.

The induction of chromosome damage in Chinese hamster (line CHO) cells by 4'-[(9-acridinyl)-amino]methanesulphon-m-anisidide-HCl (MAC) (NSC-141549) was studied in cell populations growing exponentially and at various stages of the cell cycle following release from isoleucine-deficient G1-arrest. Autoradiographic analysis demonstrated that cells in S-phase at time of drug addition (2 microgram MAC/ml for 2 hr) were delayed 8 hours before entering mitosis. Cells in G1 at the time of MAC treatment were not as severely delayed, which resulted in a rather sharp increase and decrease in a percent labeled mitosis curve. Chromosome damage occurred differentially during the cell cycle. Cells in late G2 during MAC treatment contained incompletely condensed chromosomes with occasional chromosome interchanges at the next mitosis. Early G2 cells were severely damaged (greater than 20 breaks/cell). Damage to cells in S or G1 at the time of MAC addition was less severe, whereas cells in G1-S traverse had intermediate levels of chromosome breaks. Thus MAC appeared to be particularly effective at times when chromatin was undergoing structural modifications (G1-S and S-G2 boundaries). Low concentrations of MAC (0.05 microgram/ml) increased the rate of sister chromatid exchange to almost eight times the background rate. The cellular effects of MAC were compared with previously reported studies of other antitumor agents.

Comparative effects of three nitrosourea derivatives on mammalian cell cycle progression.

Three nitrosourea analogs, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and 1-trans-(2-chloroethyl)-3-cyclohexyl-1-(4-methylcyclohexyl)-1-nitrosourea, were examined for effects on survival and cell cycle traverse capacity in exponentially growing (cycling) populations of line CHO Chinese hamster cells and in cultures arrested in G1 by isoleucine deprivation during treatment with drugs, then returned to the cycling mode by restoration of isoleucine (noncycling cells). Among parameters studied were survival, cell division, DNA initiation capacities, cell cycle distributions, and rates of cell cycle traverse in drug-treated cycling and noncycling cells utilizing a protocol combining autoradiography, cell number enumeration, and flow microfluorometry. The results obtained were in generally good agreement with results obtained in vivo in other studies and included the following. Cells treated with any of these agents accumulated preferentially in late S and G2, primarily the result of a gross increase in duration of these phases of the cell cycle. There was also a prolongation of doubling time during the early stages following drug treatment and return to the proliferating mode of cells which ultimately survived. All three drugs induced mitotic nondisjunction in cells capable of dividing and also induced polyploidy by allowing multiple rounds of progression through the cell cycle in the absence of an intervening cell division. In treated populations, the G2-arrested and polyploid cells were among the first cells to die. Treated, noncycling cells that were returned to cycle exhibited a lower survival capacity than did treated, cycling cells. Finally, 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea and 1-trans(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea induced a dramatic alteration in clonal morphology and growth patterns in surviving cells that persisted for at least a week after drug removal. The results obtained suggest that our model system may be useful as a predictive guide for determining response of susceptible tumor cells to treatment with chemotherapeutic agents.

Differential response of cultured human normal and tumor cells to trace element-induced resistance to the alkylating agent melphalan.

Previous studies using cultured Chinese hamster cells indicated that pretreatment of the cells with the trace elements copper, selenium, and/or zinc resulted in increased survival of the metal-induced cultures following subsequent exposure to mono- and bifunctional alkylating agents. To ascertain whether a comparable protective response could be activated in human-derived material, a series of human normal and tumor cells was treated with these trace elements and later challenged with the alkylating agent melphalan, prior to determination of the surviving fraction via colony formation. Normal human cells derived from either newborn infants or adults exhibited an increase in survival of 7- to 9-fold when pretreated with zinc alone that increased to approximately 16-fold when these normal cells were induced with all three trace elements. In contrast, comparable pretreatment of tumor cell populations resulted in an increase in survival of 1.7-fold or less, with most types of tumors exhibiting no induced protection. These observations describing a differential inducibility of normal and tumor cells raise the possibility of a novel approach for selectively sparing normal tissue in patients undergoing treatment with alkylating agents. Possible ramifications to cancer chemotherapy are discussed.

Flow cytometric localization within the cell cycle and isolation of viable cells following exposure to cytotoxic agents.

Viable cell sorting, based on flow cytometric analysis of DNA content and cell volume, was used to evaluate the cycle position and survival potential of Adriamycin (AdR)-treated or 1-beta-D-arabinofuranosylcytosine (ara-C)-treated CHO cells. Drug-treated cells initially stained with the vital, DNA-specific fluorochrome, Hoechst 33342, were analyzed for DNA content and volume, and sorting "windows" were established for subsequent sorting of duplicate unstained cell samples based only on cell volume. Another portion of the cell sample was fixed in ethanol, and stained with three fluorochromes for correlated flow cytometric analysis of DNA, RNA, and protein. Similarities in the viable cell volume distributions and the protein content distributions of the ethanol-fixed samples provided a means for indirectly determining the DNA and RNA contents of the sorted cells. Three regions (S, L, and I) were selected in the cell volume distributions corresponding to the range of near normal cell size (S), larger than normal cell size (I), and the extremely large cells (L). Adriamycin-treated or ara-C-treated cells sorted from the S region had survival values, respectively, 46 times and 7 times greater than the abnormally large cells in region L. Cells from the S region also respectively survived 14-fold (AdR-treated) and 7-fold (ara-C-treated) greater than the cells sorted from the I regions. RNA content levels for cells within the L region were three times and two times greater, respectively, than the AdR-treated and Ara-C-treated subpopulations in the S regions. Survival of subpopulations of G2-arrested, AdR-treated cells (I and L regions) was better correlated with relative abnormality in cell size than with position in the cell cycle. In addition to providing further support for the validity of the "balanced growth hypothesis," the results of this study suggest that two-parameter DNA content and cell volume measurements would be extremely useful for providing general guidelines for judging the effectiveness of therapy, especially in clinical diagnoses where cell sorting is impractical or impossible. From these analyses the frequency and cycle position of cells resistant to therapy can be estimated. Such information would be particularly useful for rapidly detecting drug-resistant cells and design of subsequent therapeutic regimens.

Zinc-induced resistance to alkylating agent toxicity.

Suspension cultures of Chinese hamster ovary cells and three derived cadmium-resistant variants were exposed to 100 microM ZnCl2 prior to treatment with the alkylating agent, melphalan, and cytotoxicity was then determined by measuring colony-forming ability. A 10-fold or greater enhancement in survival of all zinc-pretreated cultures subsequently exposed to melphalan was observed which was unrelated to metallothionein induction capacity. Although the maximum achievable protection afforded by zinc occurred in cultures receiving 100 microM ZnCl2, concentrations of zinc only slightly in excess of levels found in human serum were shown to provide a 4.5-fold enhancement of protection, indicating that the phenomenon can also be induced at physiologically reasonable levels. These results suggest the existence of a novel zinc-inducible mechanism which protects cells against the toxic effects of alkylating agents.

Action of heparin on mammalian nuclei. II. Cell-cycle-specific changes in chromatin organization correlate temporally with histone H1 phosphorylation.

The interaction of the polyanion heparin with the inner histones of chromatin has been used to detect changes in chromatin organization associated with cell-cycle traverse. Synchronized populations of Chinese hamster cells were obtained either in early G1 or near the G1/S boundary. The rate of interaction of heparin with chromatin-associated inner histones was measured using nuclei isolated from synchronized cell populations in different phases of the cell cycle. A G1-specific decrease in rate of interaction of heparin with inner histones was observed and found to be independent of the presence of hydroxyurea during traverse of G1. A further decrease in heparin-inner histone interaction occurred in late S and G2. These changes correlate temporally with the interphase phosphorylation(s) of histone H1. This correlation is discussed within the framework of current models of higher order chromatin structure (i.e. organization above the nucleosome level). Analysis of the cooperativity of interaction of heparin with inner histones was performed using the kinetic analog of the Hill equation. This analysis suggests that the organization of inner histones on chromatin does not undergo large variations during the cell cycle.

Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification.

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). During the time that synchronized G1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.

Action of heparin on mammalian nuclei. I. Differential extraction of histone H1 and cooperative removal of histones from chromatin.

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.