Structure-activity relations of S-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells

SAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from Ciba-Geigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 microM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the pi electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 microM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormone-responsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.     

4-Amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) exerts in vitro growth inhibitory effects that are not only related to S-adenosylmethionine decarboxylase (SAMdc) inhibition

The competitive S-adenosylmethionine decarboxylase (SAMdc; EC 4.1.1.50) inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) inhibits growth more effectively than the irreversible SAMdc inhibitor 5'-[[(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo), while having similar effects on polyamine contents. We hypothesized that growth inhibition by CGP 48664A is not merely accomplished by SAMdc inhibition. Concentration-related growth inhibitory effects of AbeAdo, CGP 48664A and methylglyoxal bis(guanylhydrazone) (MGBG) were investigated in L1210 cells that were additionally exposed to 10 microM AbeAdo. This concentration causes maximal growth inhibition, profound SAMdc inhibition and plateau polyamine contents. Almost complete inhibition of functional SAMdc activity by 10 microM AbeAdo was confirmed by demonstration of poor conversion of tetradeuterated spermidine to tetradeuterated spermine by gas chromatography-mass spectrometry. Increasing AbeAdo did not affect L1210 cell numbers, viability, nor polyamine contents. MGBG proved highly toxic. CGP 48664A did not affect L1210 polyamine contents, but cell numbers and viability decreased dose-dependently to 50% and 70% of control, respectively. We conclude that CGP 48664A inhibits L1210 growth not only through SAMdc inhibition, but also by an as yet poorly understood second effect with higher IC50. The alleged second effect of CGP 48664A appears important for its potent antitumor effect.     

Effects of lithium on cAMP-dependent protein kinase in rat brain

The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.     

Kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth

The kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth were studied. The steady deceleration of the specific growth rate with increasing tumor mass that was observed was attributable to a prolongation of the cell cycle, particularly of the S and G2 phases. The cell cycle time (Tc) was 43.3 hr (TG1 equals 10.8, TS equals 26.8, and TG2 equals 5.7 hr) on the seventh day of growth and 76.0 hr (TG1 equals 14.0, TS equals 52.0, and TG2 equals 10.0 hr) on the tenth day of growth. The growth fraction showed a decrease from 0.77 to 0.60 during the 7- to 10-day tumor growth interval. The cell death rate remained low and essentially unchanged during this period. A high correlation was found between polyamine synthesis (ornithine decarboxylase activity) and the specific growth rate; the correlation coefficient was 0.985. There was also a high positive correlation between the cellular polyamine (spermidine and spermine) and nucleic acid content (spermidine: DNA equals 0.916, spermine: DNA equals 0.947, spermidine:RNA equals 0.907, and spermine: RNA equals 0.881). These observations suggest that there may be a functional coupling between polyamines and nucleic acids, and they support the hypothesis that polyamines play an important role in DNA replication and cell division.     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd) -pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G2, i.e., the fraction of BrdUrd-negative G2 cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G2 and in S are equally delayed. S phase transit of cells irradiated in S or in G1 did not appear to be affected. There were apparent changes in flow of cells out of G1, which could be explained by the delayed entry of G2 cells into the compartment because of G2 arrest. Thus, in asynchronous cells the method was able to detect G2 delay in those cells irradiated in S and G2 phases and demonstrate the absence of cell-cycle delays in other phases.     

Cell cycle-dependent tumor necrosis factor apoptosis

To determine if tumor necrosis factor (TNF)-mediated apoptosis affects cells at defined stages of the cell cycle, WEHI-164/2F (WEHI) cells were synchronized at G0-G1 after 3-day cultures in medium containing RPMI 1640 and 0.5% FCS (RPMI-0.5% FCS). The arrested WEHI cells (60-75% in G0-G1) showed increased sensitivity to TNF killing, measured as 48-h 3-(5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, and 15-h apoptosis by propidium iodide staining and flow cytometry analysis. The TNF killing kinetics of G0-G1-arrested cells was similar to controls, and TNF did not accelerate or retard cell cycle progression of the arrested cells after feeding with fresh RPMI-0.5% FCS. However, TNF inhibited WEHI DNA synthesis as early as 1 h after treatment, and inhibition was proportionate to sensitivity to TNF-induced apoptosis. WEHI cells treated with TNF showed a higher percentage of cells in S phase with concomitant decrease in G0-G1 and G2-M. When cultured for 3-18 h in fresh RPMI-0.5% FCS to allow progression of the G0-G1-arrested cells toward the G1-S boundary, WEHI cells became more sensitive to TNF killing, especially at the 3-9 h time points. Moreover, TNF did not degrade [125I]5-iodo-2'-deoxyuridine-labeled WEHI DNA if the labeled cells were precultured for 9 h in fresh RPMI-0.5% FCS to allow them to pass S phase before the addition of TNF. These results show that TNF-induced apoptosis of WEHI cells is connected to cell cycle events; WEHI targets receive the TNF cytotoxic signal mainly at the G1-S boundary and begin to die by apoptosis as they exit from S phase.     

The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phases during hypoxic stress

We have studied the role of the retinoblastoma susceptibility gene product (pRB) in the regulation of cell-cycle progression under extremely hypoxic conditions (< 4 ppm O2). pRB is a nuclear matrix-associated phosphoprotein that normally exerts its growth-regulatory effects during early-G1 phase of the cell cycle, where all pRB present has been assumed to be in the under-phosphorylated form and bound in the nucleus. The effect of hypoxia on pRB nuclear binding and its state of phosphorylation was studied by two methods: (a) two-parametric flow cytometric measurement of pRB versus DNA and (b) Western blotting. Pulse-chase and pulse labeling with BrdUrd was used to record cell-cycle progression under versus after extremely hypoxic conditions. We demonstrate that pRB is dephosphorylated and rebound in the nucleus in more than 90% of cells located in S and G2 under extremely hypoxic conditions. While inhibition of DNA synthesis was instantaneous under hypoxic conditions, dephosphorylation and rebinding to nuclear structures of pRB takes more than 4 h. Within this time span, cells in G2 complete mitosis and divide. The slow dephosphorylation of pRB indicates that pRB is neither associated with the instantaneous inhibition of DNA synthesis nor is it the cause of the oxygen-dependent restriction point located in late-G1. The observed dephosphorylation of pRB is not dependent on functional p53, suggesting that at least one of the mechanisms responsible for the dephosphorylation is due to hypoxic activation of a pRB-specific phosphatase in the absence of p53-dependent inhibition of pRB kinase activity. However, it cannot be ruled out the participation of pRB kinase inhibitors independent of p53 activation. Cells arrested in G1 during prolonged hypoxia resumed cell-cycle progression within 2-->24 h after reoxygenation, while cells arrested in S were unable to reenter cell-cycle progression after reoxygenation. The hypoxia-induced dephosphorylation of pRB was only partly reversible by reoxygenation. Reentry into the cell cycle induced by reoxygenation occurred concomitant with unbinding (hyperphosphorylation) of pRB. Thus, rephosphorylation of pRB seem to be the rate-limiting step for reentry into the cell cycle after reoxygenation. Although pRB seems to play a major role in suppression of cell growth under and following hypoxic stress, other factors seem to be responsible for the immediate hypoxia-induced arrest in late-G1 and S phases.     

Geminin, an inhibitor of DNA replication, is degraded during mitosis

We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). We propose that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.     

Effects of recombinant human transforming growth factor-beta 1 or/and interleukin-6 on growth inhibition and proto-oncogene c-myc expression in human leukemia cells

OBJECTIVE: To examine the effect of recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) alone or recombinant human interleukin 6 (rhIL-6) alone or in combination on proliferation inhibition of the human leukaemia cell line. METHODS: In the present study, using the human monoblastic cell line (U937) and human promyelocytic cell line (HL60) as an in vitro model, we analyzed the effect of two cytokins on proliferation inhibition with rate of 3H-TdR incorporation, the cellular content of DNA, DNA indices, the cell cycle and the expression of c-myc mRNA. RESULTS: With administration of rhTGF-beta 1 and rhIL-6, U937 cell growth was inhibited and the rate of 3H-TdR incorporation inhibition was increased. There was a decrease in the cellular content of DNA and DNA indices. And no change in the cell cycle was observed after administration of rhTGF-beta 1 or rhIL-6. However, there was an increase in G0/G1 phase cells and a decrease in G2M + S phase cells after administration of combination of rhTGF-beta 1 and rhIL-6. It was also found that rhIL-6 could inhibit proliferative responses of HL60 cells, meanwhile the inhibition could be enhanced by rhTGF-beta 1. The rate of 3H-TdR incorporation inhibition rose up to 39.89%, and DNA index fell to 1.00 following induction by rhIL-6 plus rhTGF-beta 1. Furthermore, G0/G1 phase cells increased while G2M + S cells decreased. CONCLUSIONS: These results suggest that combination of rhTGF-beta 1 and rhIL-6 acted in synergy to inhibit proliferation of both U937 and HL60 cell lines. Molecular hybridization test show that rhTGF-beta 1 alone, rhIL-6 alone or rhTGF-beta 1 and rhIL-6 in combination can inhibit U937 and HL60 cells expression of c-myc mRNA in a time and dose dependent manner. rhTGF-beta 1 and rhIL-6 in combination synergistically inhibited c-myc expression, which may be one of the machanisms for the actions of the two cytokines.     

Differential expression of SHP2, a protein-tyrosine phosphatase with SRC homology-2 domains, in various types of renal tumour

The synthesis of DNA was studied in the proximal tibial growth plate of 25-day-old healthy NMRI mice by using the thymidine analog bromodeoxyuridine (BrdUrd), which is incorporated into cells in the S-phase. Such cells were found only in the upper three fifths of the morphologically defined proliferating zone. This zone was therefore subdivided into a functional proliferating zone (the S-phase zone) where most, if not all, chondrocytes proliferate, and a remaining maturation zone. The BrdUrd containing immunoreactive cells could then be followed at different intervals and they were found at the chondro-osseous junction after only 36 h. By using double-labeling with BrdUrd and iododeoxyuridine (IdUrd) the duration of cell cycle components could be estimated; that is, the time for DNA synthesis (S-phase), second gap and mitosis (G2 + M-phase), and remaining first gap (G1). We determined an S-phase time of 7.1 h and an average cell-cycle duration of 36 h. The G2 + M-phase was estimated as 3.5-4 h, leaving an average G1-phase time of 25 h, which probably varies considerably between chondrocytes. By combining these data with morphometrical data regarding distances between cells, we calculated a total growth rate of 9.0 microm/h. Of this rate, 80% was entirely related to the process of hypertrophy--that is, longitudinal expansion without any corresponding increase in cell number--and 75 % was the result of processes outside the S-phase zone. Five percent of the growth was due to the expansion of cell distances within the S-phase zone. In this way longitudinal expansion can be studied at different levels in the growth plate and the data permit calculation of changes in volumes of the extracellular matrix. The largest increases in matrix volume occurred in the hypertrophic zone. These data may serve as a basis for further studies on matrix turnover in relation to growth.     

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells

We studied the effect of doxorubicin (Dox) on cell cycle progression and its correlation with DNA damage and cytotoxicity in p53-mutant P388 cells. P388 cells synchronized in S and G2/M phases were > 3-fold more sensitive to Dox than were cells in G1 phase (Dox ID50 = 0.50 +/- 0.16 microM in cells synchronized in S phase versus 1.64 +/- 0.12 microM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 microM Dox (2 x ID50) for 1 hr induced a marked cell arrest at G2/M phase at 6-12 hr after drug incubation. We then studied the effect of Dox on the p34cdc2/cyclin B1 complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34cdc2 kinase localizes in the nucleus and cytoplasms, particularly in the centrosomes, and p34cdc2 kinase activity is dependent on cell cycle progression, with the enzyme activity increasing steadily from G1/S to G2/M and markedly declining thereafter. Treatment of synchronized P388 cells in early S phase with 1 microM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for > or = 10 hr. In a cell-free system, Dox did not inhibit p34cdc2 kinase activity, indicating that is has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34cdc2 kinase dephosphorylation without altering its synthesis, and this effect was due to neither down-regulation of cdc25C nor inhibition of protein-tyrosine phosphatase activity. In contrast, Dox treatment was found to induced cyclin B1 accumulation as a result of stimulating its synthesis and inhibiting its degradation. A good correlation was found between extent of DNA double-strand breaks and p34cdc2 kinase activity inhibition. Our results suggest that anthracycline-induced cytotoxicity is cell cycle dependent and is mediated, at least in part, by disturbance of the regulation of p34cdc2/cyclin B1 complex, thus leading to G2/M phase arrest.     

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.     

A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development

Effects of etoposide (VP-16) and cytosine arabinoside (Ara-C) on the cell cycle of HL-60 and THP-1 cells were studied by flow cytometry using the bromodeoxyuridine (BrdU)/DNA assay technique to investigate the efficacy of VP-16 for monocytic leukemia cells. VP-16 inhibited the proliferation of THP-1 cells more strongly than that of HL-60 cells at any concentrations used at 24 and 48 hr. VP-16 arrested HL-60 and THP-1 cells in the G2/M phase and reduced them in the G0/G1 and early S phase at higher concentrations. There was no significant difference in the percentage of G2/M phase cells at the same concentration between both cells. However, reduction in the G0/G1 and early S phase cells was more marked in THP-1 than HL-60 cells significantly. On the other hand, Ara-C perturbed the cell cycle of HL-60 cells more than that of THP-1 cells at 24 and 48 hr. These results suggest that the effects of VP-16 on the cell cycle may be more intense in THP-1 than HL-60 cells, and support the efficacy of VP-16 for treating monocytic leukemia in vivo.     

Cellular changes of rat embryonic fibroblasts by an actin-polymerization inhibitor, bistheonellide A, from a marine sponge

Bistheonellide A, an inhibitor of actin polymerization from the marine sponge Theonella sp., was introduced at a concentration of 100 nM into rat fibroblast of 2.4 x 10(4) cells/ml. Within 1 h, it disrupted stress fibers, accompanied by a marked change of the cell morphology, resulting in the formation of processes from the cell surface. Further incubation for 24 h in the presence of 100 nM bistheonellide A led to binucleation in most cells and subsequent inhibition of cell cycle progression. When bistheonellide A was withdrawn from the culture medium, binuclear cells began to grow again within 20 h and reverted to mononuclear morphology. Flow cytometric analysis fluorescence-activated cell sorting showed that 2C diploid DNA content in G1 phase was changed into 4C content of tetraploid for the bistheonellide A treated-cells in G1 phase and into 8C content during G2 and M phase. Therefore, we suggested that the bistheonellide A treatment inhibited cytokinesis, but not mitosis in M phase, and that treated cells were arrested at the early G1 phase. These effects of bistheonellide A on the cell cycle progression of 3Y1 fibroblast were also observed more prominently in cells synchronized in S phase with hydroxyurea. Cells in G0 phase were then activated by the addition of fetal calf serum in the presence of 100 nM bistheonellide A. Cell cycle progression of the bistheonellide A-treated cells was obviously slowed down or completely inhibited during G1 phase. These results reveal that actin filaments are not only essential to cytokinesis but also for promoting the progression of cell cycle G1 to S phase.     

IL-2-dependent induction of G1 cyclins in primary T cells is not blocked by rapamycin or cyclosporin A

Autocrine stimulation of the IL-2 receptor (IL-2R) is required for commitment of a T cell to enter the cell cycle and may involve transmission of the IL-2R signal to cell cycle control proteins. Candidates for such proteins are the D-type cyclins which are expressed in G1. Short-term cultures of primary human T cells were used to show that expression of cyclins D2 and D3 is regulated by IL-2 in a concentration- and time-dependent manner. Cyclin D2 RNA was induced rapidly to peak levels well before initiation of DNA synthesis and gradually declined during the remainder of G1. Cyclin D3 RNA and protein showed a slower induction during G1 to maximal levels as cells initiated DNA synthesis that remained high throughout S phase. Induction of cyclins D2 and D3 was independent of the cyclosporin A-sensitive calcineurin pathway and of rapamycin-sensitive pathways, despite the ability of rapamycin to severely inhibit entry into S phase. These observations suggest that cyclins D2 and D3 may monitor the IL-2R signal but that their induction does not guarantee entry into S phase. Rapamycin was found to target a pathway late in G1 that is distal to induction of D-type cyclin gene expression but proximal to DNA replication, perhaps involving the function of the D-type cyclin proteins or their associated kinases.