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.     

Cell cycle analysis of sodium butyrate and hydroxyurea, inducers of ectopic hormone production in HeLa cells

Sodium butyrate and hydroxyurea, effective inhibitors of DNA synthesis in HeLa cells, cause these cells to produce increased levels of the ectopic glycopeptide hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and free alpha chains for these hormones. The objective of this study was an assessment of the role of modulation of cell cycle events in the action of these two chemical agents. A variety of experimental approaches was employed to obtain a clear view of the drugs' effects on cells located initially in all phases of the cell cycle. Cells in early G1, G2, or M phase at time of addition of either inhibitor were not arrested at early time points, but by 48 hours became collected at a location characteristic for each drug, near the G1-S phase boundary. Flow microfluorometry (FMF) and thymidine labeling index revealed that butyrate-treated cells arrested late in G1 phase very close to S phase, while hydroxyurea-blocked cells continued to early S phase. Both inhibitors prevented cells originally in S phase from reaching mitosis. S cells exposed to hydroxyurea were killed by 48 hours, but those growing in 5 mM butyrate progressed to the end of S or G2 phase where they became irreversibly arrested although not removed from the monolayer. Analysis of the cell cycle location and viability of each subpopulation resulting from 48 hour exposure to butyrate or hydroxyurea is important for the study of the function of each cellular subset. Treatment of HeLa cells with lower concentrations of butyrate (1 mM) resulted in slowed yet exponential growth. Fraction labeled mitosis (FLM) analysis shows that this is a result of prolongation of the G1 phase.     

Dose dependent effects on cell cycle checkpoints and DNA repair by bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 μM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.     

Lethal response of HeLa cells to x-irradiation in the latter part of the generation cycle.

The age-response for the killing of HeLa S3 cells by X-rays during the latter part of the generation cycle has been examined in detail. As synchronous cells move from the G1/S boundary through S phase, the relatively high sensitivity of late G1 cells gradually decreases; minimum sensitivity is reached in mid-S and maintained during the remainder of that phase. The response of cells as they progress from S to the point in G2 at which they are temporarily arrested by radiation (or by inhibitors of protein synthesis) was measured in populations free of both S phase cells and late G2 cells that had passed the arrest point: cells retain their high resistance from early G2 up to the arrest point. The response of G2 cells that have passed the arrest point before being irradiated was examined by exposing randomly growing cultures to X-rays and collecting cells periodically thereafter, as they entered mitosis. Survival values very close to those of sensitive mitotic cells were found in the 2 h period after irradiation during which unarrested cells continued to reach mitosis. Values typical of lateS/early G2 were found only after cells that had been arrested began arriving at mitosis. Thus, HeLa S3 cell undergo an abrupt increase in sensitivity at or near the arrest point. The sensitivity to a second irradiation of cells arrested in G2 by a conditioning X-ray dose increases rapidly in the early part of the arrest period.     

Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts

When randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

The pleiotropic effects of 33258-Hoechst on the cell cycle in Chinese hamster cells in vitro

The fluorochrome 33258-Hoechst which binds to double-stranded DNA (dsDNA) has been previously shown to inhibit in several mammalian cell cultures the condensation of chromosomes in phase G2 and early mitosis. We have now found that this drug affects the cell cycle of Chinese hamster cells grown in vitro in several other ways. In cells treated with the drug, phase G2 is prolonged, the rate of DNA replication is drastically reduced and the cells are arrested most probably at very late S phase.     

Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells

Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20- 24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light- chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.     

Effects of serum deprivation on the initiation of DNA synthesis in the second generation in rat 3Y1 cells

Rat 3Y1 cells arrested at early S by hydroxyurea traversed the remainder of S and G2 and completed mitosis after removal of the drug, irrespective of the absence of serum from the culture medium. When cells were deprived of serum for a period between early S and mitosis after removal of hydroxyurea, the cells delayed entry into S in the presence of serium in the second generation for the time length approximately equal to that of serum deprivation. When mitotic cells, which had been continously exposed to serum after removal of hydroxyurea, were deprived of serum for the next 24 hours and then were reexposed to serum, the cells delayed entry into S for more than 24 hours (more than the time length of serum deprivation). On the other hand, the cells already deprived of serum between early S and G2 in the first generation were less delayed in entry into S after postmitotic 24-hour serum deprivation than were the cells exposed to serum between early S and G2 in the first generation. These results suggest that serum-dependent events continue to occur in the first generation for on-time entry into S in the next generation, and that these premitotic events (the potential for entry into S) decay if serum is absent for a long period of time after mitosis.     

Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1

Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.     

GC-7 cells are growth arrested by cytochalasin D at two different points in the cell cycle

GC-7 cells, a cell line from African green monkey kidney, which had been growth arrested in G0 phase by serum deprivation, entered S phase 15 h after serum stimulation. They were blocked from entering S phase in the presence of 0.6 micrograms/ml of cytochalasin D. The cells growth arrested between G0 and S phase by cytochalasin D entered S phase 6 h following the removal of the drug. The progression of S, G2, and M phases was not affected by cytochalasin D. On the other hand, when G0-arrested GC-7 cells were stimulated with serum for 23 h up to a late S/G2 phase and then cultured in the presence of cytochalasin D, or when an exponentially growing culture was treated with the drug, the cells were growth arrested at a point 15 h, not 6 h, before the next S phase. This point of growth arrest is kinetically similar to G0 phase, both occur 15 h before S phase, but is different from G0 in terms of c-fos expression after release from the block.     

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X- irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.     

Arrest of 3T3 cells in G1 phase in suspension culture.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Arrest of 3T3 cells in G1 phase in suspension culture

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Kinetics of the nuclear division cycle of Aspergillus nidulans.

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.     

Arrest of irradiated G1, S, or G2 cells at mitosis using nocodazole promotes repair of potentially lethal damage

The ability of synchronized Ehrlich ascites tumor cells, irradiated in G1, S, and G2 phases, to repair potentially lethal damage when arrested at mitosis by using 0.4 microgram/ml nocodazole, a specific inhibitor of microtubule polymerization, has been studied. Cells irradiated in these phases were found to repair potentially lethal damage at mitosis. The extent of this repair was similar to that observed for cells irradiated at the same stages in the cell cycle but allowed to repair potentially lethal damage by incubating in balanced salt solution for 6 hr after X irradiation.     

The effects of cyclosporins on the cell cycle of T-lymphoid cell lines

Cyclosporin A (CsA) is a cyclic endecapeptide of fungal origin displaying strong immunosuppressive properties. CsA and another active member of the cyclosporin (Cs) family, but not an inactive one, can interfere with the proliferation of some, but not all, T-lymphoid cell lines. Cells from Cs-sensitive lines accumulate in the G1 phase of the cell cycle. No effect is detected on the cycle of Cs-resistant lines. Both Cs-sensitive and Cs-resistant lines are arrested by another G1 blocker (actinomycin D) and DNA synthesis inhibitors (cytosine arabinoside, hydroxyurea), become multinucleated/polyploid when exposed to cytochalasin B (CB), are arrested in mitosis by colchicine and accumulate in G2 phase in the presence of Taxol. The effect of Cs is best evidenced when the drug is applied to cells which were already delayed in G1 by saturation density cultivation or serum deprivation. By the combined use of Cs and of other drugs working at a later phase of the cycle, results were obtained which suggest that the effect of Cs is either to delay very much the cells throughout the G1 phase or to arrest them at that G1 phase or at the following one. A correlation of the G1-blocking property of Cs with their immunosuppressive properties may be possible but is still speculative.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.     

Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts

Four temperature-sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8 degrees C are shifted up to 39.8 degrees C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8 degrees C. We studied the traverse through the S and G2 phases at 39.8 degrees C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8 degrees C were shifted down to 33.8 degrees C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.     

Analysis of a Chinese hamster temperature-sensitive cell cycle mutant arrested in early S phase

E36 ts24 is a temperature-sensitive cell cycle mutant which has been derived from the Chinese hamster lung cell line E36. This mutant is arrested in phase S when incubated at the restrictive temperature (40.3 degrees C) for growth. At this temperature, proliferation of the mutant cells ceases after 10 h. About 2 h earlier, DNA synthesis is arrested. These kinetic studies indicate that the execution point of the mutant cells is in early S phase well beyond the G1/S boundary. The pattern of replication bands in E36 ts24 cell grown for 9 h at 40.3 degrees C strengthen the kinetic studies and map the execution point to early S phase. The exact point of arrest of the mutant cells in phase S was mapped in early S phase near the execution point. At the point of arrest the cells continue to synthesize DNA at at a high rate but practically all of the newly synthesized DNA is degraded. This high rate of DNA degradation is limited to nascent DNA at the point of arrest. In the presence of 5-bromodeoxyuridine (5-BudR), the last E36 ts24 cells which reach mitosis at the restrictive temperature for growth show asymmetric replication bands which illustrate DNA degradation and resynthesis occurring in these cells at 40.3 degrees C.