THE ACTION OF HYDROXYUREA ON MOUSE L-CELLS*

The toxic and inhibitory properties of hydroxyurea (HU) have been studied in asynchronous and synchronized populations of mouse L-cells. Hydroxyurea is a potent growth inhibitor and appears to be specifically lethal for cells which are in the early part of S phase at the time the compound is introduced. Cells in late S phase, G₂, mitosis and G₁ appear to progress normally around the cycle in the presence of the compound until they reach the G₁/S boundary. There are indications that at least some G₁ cells are able to enter the S phase even in the presence of the drug; however their flow into S is much slower than that of control cells and therefore they are killed at a slow rate. Upon prolonged exposure to the drug a second phase of more rapid killing is observed, beginning at about the time division would occur in uninhibited cells. Hydroxyurea exhibits a rapid and marked inhibition on DNA synthesis but its effect on RNA synthesis is much less pronounced and may be a consequence of the inhibition of DNA synthesis. The effects of hydroxyurea on cell viability and DNA synthesis can be partially prevented by the addition of deoxyribonucleosides which in sufficient concentration appear to compete temporarily with the drug. The fact that the protection is only temporary would appear to rule out the hypothesis that the primary mode of action of the drug is the inhibition of the reduction which converts ribonucleotides to deoxyribonucleotides. The data presented in this communication taken together with observations of other workers would appear to suggest that the effect of the drug may be directly on the DNA molecule.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

The time and duration of the premeiotic inteprhase in microsporocytes ofTrillium kamtschaticum

The time and duration of each phase of the premeiotic interphase were determined in microsporocytes of two clones (S and K clones) ofTrillium kamtschaticum. After collectionTrillium plants were stored at 3 C or 7 C prior to completion of premeiotic mitosis in archesporial cells. For autoradiography, cells were explanted in the presence of³H-thymidine to identify the interval of the premeiotic DNA synthesis. Approximate durations of the G₁, S and G₂ phases for the K clone stored at 3 C were estimated to be 12, 12 and 14 days, respectively. The interval of premeiotic development was markedly different between clones. A high degree of synchrony in meiotic development, which is usually observed within anthers up to late meiotic prophase, was confirmed at the S phase, suggesting that synchrony is established during the G₁ interval.     

THE EFFECT OF HYDROCORTISONE ON HeLa CELL GROWTH

HeLa Chessen cells have a doubling time of 18 hr when grown in MEM containing 10% calf serum and antibiotics. When hydrocortisone (1.7 μg/ml) is added to exponentially distributed cells in log growth in this medium, a new pattern of growth begins to emerge after 10–12 hr. This pattern is characterized by a transitional state lasting for about 6 hr, and then a new doubling time of about 35 hr is maintained thereafter. Hydrocortisone removes about 5% of the cells from the proliferative pool and extends the generation time of proliferating cells to about 30 hr. The extension of the generation cycle appears to occur almost entirely in late G₁. Cells grown as clones (average 6 cells/clone) prior to the addition of hydrocortisone, undergo these changes with doses as low as 0.00017 μg/ml of medium. When the average clone size is 1.5 cells per clone, the drug concentration must be 0.017 μg/ml or higher to initiate this response. The HeLa S₃ strain continues to grow with an 18-hr doubling time in the presence of hydrocortisone after a temporary delay in growth occurring between the 12th and 16th hour.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134–144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G₁ phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G₁ phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G₁- and S-phase cells, respectively. The G₁ cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G₁ nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G₂ (or G₂-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G₂ is 30-fold greater than it is in G₁. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.     

All-trans-Retinoic Acid Blocks Cell Cycle Progression of Human Ovarian Adenocarcinoma Cells at Late G1

We prepared single cell clones from two ovarian carcinoma cell lines, CA-OV3 and SK-OV3, and analyzed the effect of all-trans-RA treatment on cell division, DNA synthesis, and cell cycle stage distribution of these single cell clones. Our results show that despite the well-known heterogeneous nature of these cell lines, all single cell clones of SK-OV3 cells are resistant to the growth inhibitory effects of all-trans-RA. In contrast, all single cell clones of CA-OV3 cells were growth inhibited by all-trans-RA. However, the extent of growth inhibition did vary somewhat from clone to clone. Additional studies employing flow cytometry showed that all-trans-RA blocked CA-OV3 cell cycle progression in the G₁stage. Finally, all-trans-RA was able to inhibit G₁progression in growth-arrested CA-OV3 cells following stimulation with fetal bovine serum, insulin, IGF-1, or estrogen. Since each of these growth factors is known to act via distinct signal transduction pathways, our results suggest that all-trans-RA blocks G₁progression by targeting a downstream process or event which occurs at a point after the insulin/IGF-1, estrogen, and serum signal transduction pathways converge.     

Apoptosis in anititumor strategies: Modulation of cell cycle or differentiation

There is a strong evidence that administration of antitumor drugs triggers apoptotic death of target cells. A characteristic feature of appotosis is active participation of the affected cell in its demise. Attempts have been made, therefore, to potentiate the cytotoxicity of a variety of agents by modulating the propensity of cells to respond by apoptosis. Several strategies to enhance apoptosis that involve modulation of the cell cycle or differentiation are discussed. Loss of control of the G₁ checkpoint in tumor cells allows one to design treatments that arrest normal cells at the checkpoint and attempt to selectively kill tumor cells with S phase specific drugs. The possibility of a restoration of the apoptosis triggering function of the tumor suppressor gene p53 when the G₁ checkpoint function is abolished is expected to increase tumor cells' sensitivity to S phase poisons. Because induction of apoptosis by many antitumor drugs is cell cycle phase specific, drug combinations that preferentially trigger apoptosis at different phases of the cycle, or recruitment of cells to the sensitive phase, offer another antitumor strategy. There is also evidence that apoptosis is potentiated when cell differentiation is triggered follwing DNA damage. This observation suggests that strategies which combine DNA damaging and differentiating drugs, under conditions where the latter are administered following DNA damage caused by the former, may be successful.     

Cell cycle kinetics of the accumulation of heavy and light chain immunoglobulin proteins in a mouse hybridoma cell line

Rates of accumulation of immunoglobulin proteins have been determined using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G₁, S and G₂+M cell cycle phases. A producer cell line that secretes monoclonal antibodies, and a nonproducer clone that synthesizes only -light chains were analyzed. The pattern for the kinetics of total intracellular antibody accumulation during the cell cycle is very similar to the previously described pattern for total protein accumulation (Kromenaker & Srienc 1991). The relative mean rate of heavy chain accumulation during the S phase was approximately half the relative mean rate of light chain accumulation during this cell cycle phase. This indicates an unbalanced synthesis of heavy and light chains that becomes most pronounced during this cell cycle phase. The nonproducer cells have on average an intracellular light chain content that is 42% lower than that of the producer cells. The nonproducer cells in the G₁ phase with low light chain content did not have a significantly higher rate of light chain accumulation relative to other G₁ phase nonproducer cells. This is in sharp contrast to what was observed for the G₁ phase producer cells. In addition, although the relative mean rate of accumulation of light chain was negative for G₂+M phase nonproducer cells, the magnitude of this relative mean rate was less than half that observed for the producer cells in this cell cycle phase. This suggests that the mechanisms that regulate the transport of fully assembled antibody molecules through the secretion pathway differ from those which regulate the secretion of free light chains. The results reported here indicate that there is a distinct pattern for the cell cycle dynamics of antibody synthesis and secretion in hybridomas. These results are consistent with a model for the dynamics of secretion which suggests that the rate of accumulation of secreted proteins will be greatest for newborn cells due to an interruption of the secretion pathway during mitosis.     

STUDY OF THE EFFECT OF HYDROXYUREA ON THE ERYTHROPOIETIN-SENSITIVE CELLS

The response of polycythaemic mice to a standard dose of erythropoietin has been measured at various, time intervals after single or repeated injections of hydroxyurea. The results exclude S phase of the cell cycle as the period responsive to erythropoietin. They suggest the existence of feedback mechanisms within the cell cycle, operating at the G₁-S boundary and within the G₁ phase. Hydroxyurea given to polycythaemic mice at various time intervals after erythropoietin induced characteristic changes in the response. These changes can be explained if both gradual transit of differentiated cells into the DNA synthesis (S phase) and changes in amount of the erythropoietin sensitive cells caused by the feedback mechanisms operating in the cell cycle are considered.     

Ploidy effect on the radiation reaction of mammalian cells

Summary The variation of X-ray sensitivity was investigated during the cell cycle. The cells were most sensitive during the S phase and less sensitive during the G₂- and G₁ phase. Furthermore, the repair of X-ray damage was investigated in stationary (plateau phase) cells. The cells of both lines were able to repair damage to nearly the same extent.

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Zusammenfassung Die Variation der Strahlensensibilität während des Zellcyclus wurde untersucht. Die Zellen reagierten am sensibelsten in der S-Phase, weniger sensibel in der G₂-und G₁-Phase. Weiterhin wurde die Reparation von Strahlenschäden bei stationären (Plateauphase-)Zellen untersucht. Die Zellen beider Linien sind in etwa gleichem Maße reparationsfähig.

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Supported (I-IV) by the Deutsche Forschungsgemeinschaft (Mi/100, 1-7).     

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro

Summary Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G₂ phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G₂ phase was apparent. However, cells in G₁ at the time of drug administration were arrested in that phase whereas those cells in S or G₂ were able to complete one mitosis before becoming arrested in the G₁ phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs. This work was supported in part by NIH Cancer Research Center Grant CA-13525 and CA-19992 from NCI, and by the Association for Brain Tumor Research. Presented at the 6th International Cell Cycle Conference, March, 1976, New Orleans, Louisiana. The tumor used in this study was provided by William H. Sweet, Paul T. Kornblith, Janette L. Messer and Beverly O. Whitman of the Massachusetts General Hospital, Boston, Massachusetts.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. II. Ribosome complement in resting and early G1 cells

Using a new, sensitive and quantitative technique for determining the ribosomal-RNA content of a measured number of cells, the cellular ribosome complement was compared for cultured hamster embryo cells in the stationary growth phase and in the early G₁ phase of the cell cycle. Cells from stationary phase cultures were found to contain less than 70% of the ribosome complement of the early G₁ phase cells, though the volumes of the two cell types were similar. This would imply that the stationary phase cell is physiologically different from a cell merely arrested at some point in the cell cycle.     

EFFECTS OF IRRADIATION ON THE GENERATIVE CYCLE OF THE ESTROGEN STIMULATED VAGINAL EPITHELIUM

The effects of irradiation (300, 500 and 1500 rads) on mitosis and DNA synthesis in the estrogen primed vaginal epithelium have been studied. Dose-effect relations and the time sequence of effects on the two processes were investigated. The technique of tritiated thymidine labeling of DNA with autoradiography was used, in conjunction with the mitotic count, to study alterations in the generative cycle. Prior to irradiation, ovariectomized female rats were treated daily with diethylstilbestrol for a period of 2 weeks to create a steady state in the vaginal cell population. It was observed that:

• 1 Within 1 hr following ionizing radiation, mitotic figures disappear from the population and reappear at a time that is dose dependent. Those cells that have begun mitosis at the time of irradiation were able to complete that phase but no cells which were in G₂ were able to begin mitosis. Therefore, a G₂ block occurs within 1 hr post-irradiation.
• 2 Radiation reduces the rate of DNA synthesis thus prolonging the S phase. There is no evidence of a radiation-induced G₁ to S block in this system.

Based on these observations, it was further hypothesized that:

• 1 Cells in G₁ at the time of irradiation are relatively insensitive and continue to progress through the generative cycle at a rate primarily determined by the level of estrogen stimulation.
• 2 Radiation may interfere with the estrogen priming mechanism in this hormonedependent system thereby reducing the effective level of estrogen stimulation. This is seen in the behavior of cells which were in G₁ at the time of irradiation. The extent of the blockage of estrogen increases with radiation dose and after 1500 rads, estrogen stimulation is essentially at castrate level.

     

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B1 expression in human melanoma cells

We have investigated the effect of mild hyperthermia (42°C) on the cytotoxic activity of a 1 h melphalan exposure in human melanoma cell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all cell lines, with a reduction in the IC₅₀ of 1.7 to 2.6-fold. Flow cytometric analysis showed that in normal temperature conditions melphalan caused S phase cell accumulation, which was evident only at 24 h in JR8, M14 and 2/21 cell lines and was still persistent at 72 h in 2/60 cells. Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G₂+ M, which was transient in JR8 and M14 cells and persisted until 72 h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melphalan induced G₂+ M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug were similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphalan cytotoxicity was observed, cell accumulation in G₂+ M was still present 120 h after treatment. The accumulation was accompanied by an inhibition in the G₂ - M transition, as demonstrated by the significant reduction in the mitotic index of cells exposed to combined treatment compared to controls. Moreover, a bivariate distribution of cells stained for DNA and cyclin B₁ showed that, following melphalan and hyperthermia treatment, the fraction of cyclin B₁-expressing cells paralleled the fraction of G₂+ M phase cells, thus indicating that the inability of cells to enter mitosis was not ascribable to a reduction of cyclin B₁ expression. On the whole, our results indicate that hyperthermia can stabilize the G₂ accumulation induced by melphalan in human melanoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation was not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.