Differential effects of ACTH or 8-Br-cAMP on growth and replication in a functional adrenal tumor cell line

The effects of ACTH and 8-Br-cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y-1) were investigated. ACTH and 8-Br-cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G₁; an additional point of arrest in G₂ occurred with 8-Br-cAMP. Cells whose growth was arrested in G₁ by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G₁ by serum deprivation. When ACTH or 8-Br-cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8-Br-cAMP had no effect on the serum-induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre-replicative period. Partial inhibition of the serum-induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH-arrested Y-1 cell resembles an in vivo hypertrophied adrenal cortical cell.     

On the regulation of DNA synthesis in a line of adrenocortical tumor cells: effect of serum, adrenocorticotropin and pituitary factors.

Y-1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y-1 cells and another active with both fibroblasts and Y-1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.     

A temperature-sensitive cell cycle mutant of the BHK cell line

A temperature-sensitive growth mutant derived from the BHK 21 cell Line, ts AF8, was found to have greatly reduced DNA synthesis at the nonpermissive temperature. This reduction is mainly due to a decrease in the frequency of cells synthesizing DNA. Upon shift up, ts AF8 becomes blocked in the G1 phase of the cell cycle. The cells acquire elevated cAMP levels and a unimodal distribution of DNA content, equivalent to that of G1 cells at the permissive temperature, Ts AF8 cells blocked at the G1/S boundary with hydroxyurea will enter S when shifted to the nonpermissive temperature. On the other hand, ts AF8 cells arrested m G1 by serum deprivation and shifted to the nonpermissive temperature at the moment of serum addition do not enter S, while those synchronized by isoleucine deprivation and shifted at the time of isoleucine addition will enter S. These data suggest that the cycle arrest point of the ts AF8 mutation is located in G1 between the blocks induced by serum starvation and isoleucine deprivation. The reduction in DNA synthesis caused by the ts AF8 mutation is not reversed by infection or transformation with Polyoma virus. Mitochondrial DNA continues to be synthesized at wild-type levels at the nonpermissive temperature.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Serum-independent regulation of initiation of DNA synthesis relating to temperature-sensitive defect in rat 3Y1tsD123 fibroblasts and its compensation by simian virus 40

Randomly proliferating 3Y1tsD123 cells are arrested in G1 phase within 24 h after a shift up to 39.8 degrees C (temperature arrest), yet the density-arrested cells (prepared at 33.8 degrees C) enter S phase at 39.8 degrees C with serum stimulation, with or without preexposure to 39.8 degrees C for 24 h (Zaitsu and Kimura 1984a). When the density-arrested 3Y1tsD123 cells were preexposed to 39.8 degrees C for 96 h, they lost the ability to enter S phase at 39.8 degrees C by serum stimulation and required a longer lag time to enter S phase at 33.8 degrees C by serum stimulation than did the cells not preexposed to 39.8 degrees C. Simian virus 40 induced cellular DNA synthesis at 39.8 degrees C in the density-arrested 3Y1tsD123 preexposed to 39.8 degrees C for 96 h. In the absence of serum after a shift down to 33.8 degrees C, the temperature-arrested 3Y1tsD123 cells entered S phase and then divided once. We postulate from these results that (1) the ts defect in 3Y1tsD123 is involved in a serum-independent process. Once this process is accomplished, its accomplishment is invalidated slowly with preexposure to 39.8 degrees C. This and the serum-dependent processes occur in parallel but not necessarily simultaneously. The accomplishment of both (all) processes is required for the initiation of S phase. The density-arrested 3Y1tsD123 cells have accomplished the serum-independent process related to the ts defect, but have not accomplished serum-dependent processes. In case of the temperature-arrested 3Y1tsD123 cells, the reverse holds true. The lag time for entry into S phase depends on the preparedness for the initiation of DNA synthesis (on the extent of accomplishment of each of all processes required for entry into S phase). (2) To induce cellular DNA synthesis, simian virus 40 stimulates directly the serum-independent process. However, we do not rule out the possibility that simian virus 40 stimulates serum-dependent processes simultaneously.     

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

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.     

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.     

Serum-mediated regulation of amino acid transport in cultured chick embryo fibroblasts

Three different temperature sensitive mutants derived from the Syrian hamster cell line BHK 21 were found to have greatly reduced DNA synthesis at the non-permissive temperature. These mutants are distinct by complementation analysis and behave at the non-permissive temperature as cell cycle traverse defective mutants. Microfluorometric analysis of mutant populations arrested at the non-permissive temperature shows an accumulation of cells with G1 DNA content. Mutants ts 13 and ts HJ4 synchronized in G1 by serum or isoleucine deprivation and shifted to the non-permissive temperature at the time of release do not enter the S phase, while in the case of mutant ts 11 preincubation at the non-permissive temperature before release is required to completely prevent its entry into S. Ts 13 and ts 11 are able to traverse the S phase at the non-permissive temperature when synchronized at the boundary G1/S; in this case, preincubation of ts 11 at the non-permissive temperature before release does not affect the ability of these cells to perform DNA synthesis. On the other hand, ts HJ4 appears to traverse S only partially when tested under similar conditions. Temperature shift experiments of mutant populations at different times after isoleucine synchronization suggest that ts 13 and ts 11 are blocked at the non-permissive temperature in early G1, whereas ts HJ4 is probably affected near the initiation of DNA synthesis, or in some early S function.     

MyoD induced cell cycle arrest is associated with increased nuclear affinity of the Rb protein.

In studying the mechanism through which the myogenic determination protein MyoD prevents entry into the S phase of the cell cycle, we have found a relationship between MyoD and the retinoblastoma (Rb) tumor suppressor protein. By direct needle microinjection of purified recombinant MyoD protein into quiescent fibroblasts, which were then induced to proliferate by serum, we found that MyoD arrested progression of the cell cycle, in agreement with studies utilizing expression constructs for MyoD. By studying temporal changes in cells injected with MyoD protein, it was found that MyoD did not prevent serum induced expression of the protooncogene c-Fos, an event that occurs in the G0 to G1 transition of the cycle. Injection of the MyoD protein as late as 8 h after the addition of serum still caused an inhibition in DNA synthesis, suggesting that MyoD inhibits the G1 to S transition as opposed to the G0 to G1 transition. MyoD injection did not prevent the expression of cyclin A. However MyoD injection did result in a block in the increase in Rb extractibility normally seen in late G1 phase cells. As this phenomenon is associated with the hyperphosphorylation of Rb at this point in the cell cycle and is correlated with progression into S phase, this provides further evidence that MyoD blocks the cycle late in G1.     

Cytoplasmic regulation of two G1-specific temperature-sensitive functions

tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state.     

Glucocorticoid-induced G1 arrest and the release effect of epidermal growth factor on the human salivary gland adenocarcinoma cell

Dexamethasone (1 microM) decreased the distribution of cells in S phase (about 75%) and increased that of G1 cells (1.1-fold) in the DNA histogram of human submandibular salivary gland adenocarcinoma cells (HSG) reversibly. In synchronized cells at G1 phase, glucocorticoid delayed the initiation of DNA synthesis by about 3-4 h. The conditioned medium (50%) or exogenous human epidermal growth factor (EGF, 10 ng/ml) significantly nullified these effects by glucocorticoids. These results suggested that glucocorticoids arrested the cells at G1 phase, which implied the inhibition of production of some progressive factor, probably EGF, in the cell cycle of HSG.     

Growth arrest of proadipocytes at a distinct predifferentiation G1 state associated with cytotoxic responsiveness to 8-bromo cyclic AMP

The control of cell proliferation can result from the coupling of growth arrest and differentiation. In this regard, we recently demonstrated that growth arrest which precedes the differentiation of 3T3 T proadipocytes must occur at a distinct state in the G1 phase of the cell cycle (GD). Cells arrested at GD differ in several biological parameters from cells arrested in G1 at other states induced by either serum deprivation (GS) or nutrient deficiency (GN). Specifically, GD-arrested cells can differentiate in the absence of DNA synthesis and GD-arrested cells can be induced to proliferate when stimulated with 1-methyl-3-isobutylxanthine; GS- and GN-arrested cells cannot. In addition, GD-, GS- and GN-arrested cells reside at topographically distinct states in G1. We now report that GD-arrested proadipocytes are also distinct in that they are highly sensitive to a cytotoxic effect of 8-bromocyclic AMP, whereas GS- and GN-arrested cells are not.     

The recovery of mammalian cells treated with methyl methanesulfonate, nitrogen mustard or UV light. I. The effect of alkylation products on DNA replication.

CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.     

12-O-tetradecanoylphorbol-13-acetate induces transient cell cycle arrest in G1 and G2 in metastatic melanoma cells: inhibition of phosphorylation of p34cdc2.

The growth of Demel human metastatic melanoma cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other nonphorbol tumor promoters including palytoxin and okadaic acid. Using flow cytometry, we have demonstrated that the cells arrested growth in G1 and G2 phases of the cell cycle. Detailed analysis of the kinetics of the growth arrest in unsynchronized cells showed that (a) the growth arrest was transient and peaked 16-20 h following addition of TPA; (b) effects of TPA on cell growth began within 1-2 h after the addition; and (c) cells completed S phase and arrested in G2. In addition, TPA induced a pronounced morphological change, which peaked by 1 h and gradually subsided over 24 h. In populations of cells synchronized in G1 using lovastatin, (a) addition of TPA blocked the onset of DNA synthesis up to the end of G1; (b) the lag between addition of the drug and onset of DNA synthesis was less than 30 min; and (c) addition of TPA at the end of G1 prevented the increased phosphorylation of p34cdc2, as determined by immunoprecipitation. The experiments reported here show that TPA transiently blocked the proliferation of Demel melanoma cells at the G1-S border and in G2, thus preventing cells from progressing through the cell cycle. These experiments suggest that pathways involving protein kinase C interact with and rapidly alter the molecular pathways involving p34cdc2 which regulate the onset of DNA synthesis and the G2-M transition.     

Serum stimulation of stationary human glia and glioma cells in culture

Stationary cell cultures can be stimulated to re-enter the cell cycle by giving medium containing fresh serum. The mechanism for this initiation was studied in normal human glia and neoplastic glioma cells in order to demonstrate any differences between normal and neoplastic cells regarding the kinetics of their march through G 1 into the S phase of the cell cycle. Glia cells were found to demand at least 12 h of serum stimulation to enter the cell cycle and were committed to DNA synthesis about 4 h prior to the actual onset of this synthesis. On the other hand, as little as l h of serum stimulation induced DNA synthesis in a fraction of glioma cells and these malignant cells were committed during the largest part of the prereplicative phase. This difference in commitment kinetics could not be explained by differences in cell cycle parameters or medium deficiencies and is considered to reflect a part of the fundamental difference between normal and neoplastic cells as regards their growth control.     

Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.