Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71-95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.     

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.     

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.     

Cyclin D2 is a moderately oscillating nucleoprotein required for G1 phase progression in specific cell types

To explore regulation and function of cyclin D2, a candidate cell cycle-regulatory proto-oncogene, we examined subcellular localisation, cell type- and cell cycle-dependent expression, and requirement of cyclin D2 protein for G1 progression, in a panel of 40 human normal and cancer cell types. Except for lymphoid cells and sarcoma cell lines, expression of cyclin D2 was considerably more restricted than that of cyclin D1, whereas both D-type cyclin proteins were low or undetectable in cells lacking functional retinoblastoma gene product. In G1 cells, the cyclin D2 protein was more resistant to extraction and localised predominantly to nuclei, whereas it became more soluble and distributed in both nuclei and cytoplasm from G1/S transition onwards. Centrifugal elutriation and multiparameter flow cytometry analyses of several cell types showed moderate cell cycle oscillation with maximum levels of the cyclin D2 protein reached in late G1. Microinjection and/or electroporation of antibodies to cyclin D2 during G1 arrested the cyclin D2-expressing lymphocytes, breast myoepithelium, and U-2-OS sarcoma cells in G1 phase, whereas cyclin D2-negative cell types were unaffected by such treatment. Consistent with the putative proto-oncogenic role of cyclin D2 in specific cell types, our data show that this G1 cyclin has properties closely resembling those of cyclin D1, including the essential positive role in regulation of G1.     

Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures.     

Yeast cells can enter a quiescent state through G1, S, G2, or M phase of the cell cycle

We have examined the ability of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae to enter a quiescent state through G1, S, G2, or M phase of the cell cycle. We monitored entry to a quiescent state by measuring two well known properties of quiescent cells, i.e., long-term viability and a dramatic increase in resistance to thermal heat shock relative to cycling cells. For this purpose, we made use of yeast cell division cycle (cdc) mutants with which we could arrest most of the cells in culture at specific points in the cell cycle. We find that these eukaryotes can enter a reversible quiescent state at any of the points in the cell cycle we examined if the cells are exposed to starvation conditions (starvation normally signals cells to leave the cell cycle). These findings indicate that mechanisms involved in entry to and exit from a quiescent state can operate not only in G1 phase (leading to G0 arrested cells) but can also operate in S, G2, and M phases of the cell cycle. These findings may be important for clinical oncology in cases where tumor cells escape the cytotoxic effects of chemotherapeutic agents. It may be that escape from the effect of these drugs is due to tumor cells entering quiescent states at points in the cell cycle other than G1 phase. Perhaps different chemotherapeutic strategies may be required to kill tumor cells reentering the cell cycle from other than G1.     

Sensitivity of human prostatic carcinoma cell lines to low dose rate radiation exposure

PURPOSE: Low dose rate radioemitters, such as 125I, 103Pd, and 89Sr, have been used both for local and systemic treatment of prostate cancer. Most normal cells exposed to ionizing radiation characteristically activate cell cycle checkpoints, resulting in cell cycle arrest at the G1/S and G2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G2/M transition point as a result of low dose rate radiation exposure appear to become sensitive to further low dose rate irradiation. For this reason, protracted exposure of cancer cells to low dose rate radiation has been proposed to result in increased cancer cell killing as compared with brief exposures of cancer cells to high dose rate radiation. Since many human prostatic carcinomas contain somatic genome alterations targeting genes which affect the cell cycle and radiation-associated cell cycle checkpoints, we evaluated the effects of low dose rate radiation exposure on the cell cycle and on clonogenic survival for various human prostatic carcinoma cell lines. MATERIALS AND METHODS: Human prostatic carcinoma cells from the LNCaP, DU 145, PC-3, PPC-1, and TSU-Pr1 cell lines were exposed to low dose rate (0.25 Gy/hour) or high dose rate (60 Gy/hour) radiation in vitro and then assessed for radiation cytotoxicity by clonogenic survival assay. Cell cycle perturbations following protracted exposure to low dose rate radiation were evaluated using flow cytometry. RESULTS: For LNCaP cells, low dose rate radiation exposure resulted in an accumulation of cells at both the G1/S and the G2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G2/M cell cycle arrest, but not G1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G1/S and G2/M cell cycle arrest versus G2/M arrest alone, appeared to have little effect on low dose rate radiation survival. Furthermore, while PC-3, PPC-1, and TSU-Pr1 cells exhibited increased cytotoxic sensitivity to low dose rate versus fractionated high dose rate radiation treatment, DU 145 and LNCaP cells did not. CONCLUSIONS: Radiation-associated pertubations in replicative cell cycle progression were not dominant determinants of low dose rate radiation killing efficacy in human prostate cancer cell lines in vitro.     

Influence of clinical status on the efficacy of stored platelet transfusion

By flow cytometric dual parameter analysis of proliferating cell nuclear antigen (PCNA) and the Ki-67 antigen a detailed cell cycle analysis can be performed. In this study the coordinated expression of these two growth-related antigens was investigated in human haematopoietic cells at entrance into the cell cycle as well as at exit from the cycle. In mitogen-stimulated peripheral blood lymphocytes entering the first cell cycle, the Ki-67 antigen was found to be expressed in S phase cells and not in G1 cells. Thus, the Ki-67 antigen expression in PCNA-positive S phase cells differed between continuously cycling cells and cells entering the cell cycle. Based on this difference, it was possible to visualize and evaluate the recruitment of cells into the first cell cycle from a resting stage. This new cell cycle parameter can give additional information concerning tumour growth. The Ki-67 antigen was also studied during different stages of G1 and was found to be expressed at high levels in early G1 cells compared with other parts of G1.     

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.     

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine

Protoplasts isolated from petunia leaf mesophyll are non-cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cdc2 kinase activity to cell cycle phase. Our results show that the histone H1 phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at G1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative p34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.     

Ordered cell cycle phase perturbations in Chinese hamster ovary cells treated with an S-adenosylmethionine decarboxylase inhibitor

The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine-depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd-containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd-free medium before sampling (post-labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti-BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd-nonlabelled G1 cells into S phase during the post-labelling time, a measure of the G1/S transition was obtained. The time of appearence in G1 of BrdUrd-labelled cells which had divided was used to monitor the length of the G2+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd-labelled cells through S phase. CGP 48664-induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects on the G1/S transition or on the length of the G2+M phase were observed until 2 days after seeding.     

G1 arrest of U937 cells by onconase is associated with suppression of cyclin D3 expression, induction of p16INK4A, p21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation

Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.     

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.     

Population dynamics of the monogenean Anoplodiscus cirrusspiralis on the snapper, Pagrus auratus

Mobilized peripheral blood progenitor cells (PBPC) have been shown to differ qualitatively from bone marrow (BM) progenitors. The released progenitor cells are predominantly in G0/G1 and show a relatively high percentage of rhodamine dull cells. Within the BM these last two features are characteristic of the more primitive progenitors. Although the mobilized PB cells can give rise to long-term repopulation and thus contain stem cells, the frequency of stem cells is not much higher if long-term initiating cell (LTC-IC) assays are used. To determine whether quiescent stem cells are selectively released or the low-cycle status of PB progenitors is related to the release from the BM microenvironment, the cell cycle status and rhodamine content in the PB and BM during mobilization were studied and compared with steady-state BM. More differentiated and more primitive progenitors were separated based on differentiation markers and cloned in single cell assay. In mobilized PB 54% of the CD34+ cells (n=5) were rhodamine dull compared to 22% in steady-state BM (P=0.014) [n=6]. The percentage of CD34+ cells in the S/G2M phases of the cell cycle was 2.1% in the mobilized PB (n=11), and 18% in steady-state BM (n=11) [P=0.002]. During mobilization the fraction of cells in the S/G2M phase of the cell cycle was 16% in BM (n=7), similar to steady-state BM (P=0.34). The released progenitors represented a selection of BM progenitors, with significantly more primitive progenitors (CD34+/13+/33dim) and less lymphoid precursors (CD34+/19+). Within the more differentiated CD34+113+/33bright, myelomonocytic precursors, both in PB as well as in BM, the percentage S/G2M was relatively higher than in the CD34+/13+/33dim subfraction: in normal BM: median 18% vs 8% (P=0.006) [n=8]; in mobilized PB 3% vs 2% (P=0.03) [n=10]; and in BM during mobilization 24% vs 7% (P=0.01) [n=6]. The cycle status of mobilized PB progenitors was low both in the primitive and more differentiated subfractions. During the mobilization period the BM progenitors are cycling as in steady-state BM. The low-cycle status of the mobilized PB progenitors may be related to the loss of contact with the micro-environment.     

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.     

Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells

Flavopiridol (NSC 649890, L86-8275), a potent inhibitor of cyclin-dependent kinase 1/p34cdc2 phosphorylation and kinase activity, is currently undergoing Phase I clinical testing as a potential antineoplastic agent. Previous studies have suggested that flavopiridol is cytostatic but not cytotoxic when applied to exponentially growing cells. In the present study, various human tumor cell lines were assayed for trypan blue exclusion and ability to form colonies after exposure to flavopiridol under a variety of growth conditions. When log phase A549 non-small cell lung cancer cells were examined 72 h after the start of a 24-h flavopiridol exposure, as many as 90% of the cells accumulated trypan blue. A 24-h exposure to 250-300 nM resulted in trypan blue uptake in 50% of A549 cells at 72 h and a 50% reduction in colony formation. Similar results were observed in HCT8 ileocecal adenocarcinoma, T98G glioblastoma, MCF-7 breast adenocarcinoma, and HL-60 leukemia cells. With A549 cells, identical results were obtained in actively growing logarithmic phase cells and growth-arrested confluent cells. Treatment with the DNA synthesis inhibitor aphidicolin only minimally affected the cytotoxicity of flavopiridol. In contrast, the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the protein synthesis inhibitor cycloheximide reduced the cytotoxicity of flavopiridol. These results suggest that: (a) flavopiridol is not only cytostatic, but also cytotoxic to a variety of human tumor cell lines; (b) flavopiridol is equally active against cycling and noncycling A549 cells; and (c) RNA and protein synthesis appear to play a role in flavopiridol-induced cytotoxicity.     

Expression of wild-type p53 increases etoposide cytotoxicity in M1 myeloid leukemia cells by facilitated G2 to M transition: implications for gene therapy

We have evaluated the role of p53 in the induction of cell death by the DNA topoisomerase II inhibitor etoposide in M1 myeloid leukemia cells. Three different clones of M1 cells were used: S6, which lacks p53; Phe-132, which expresses mutant p53 constitutively; and LTR-13, which expresses mutant protein at 37 degrees C and wild-type p53 at 32 degrees C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32 degrees C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 microg/ml) of all three cell lines at 37 degrees C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferentially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss of cells in late S and G2-M phase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type p53 and etoposide) leads to suppression of the etoposide-induced G2 block. To determine the effect of p53 on the G2 to M transition, LTR-13 cells were incubated with etoposide for 24 h at 37 degrees C and then either maintained for an additional 12 h at 37 degrees C or shifted to 32 degrees C to activate wild-type p53. The expression of wild-type p53 resulted in an increase in mitosis-specific phosphorylation, as determined by the MPM-2 antibody as well as the formation of mitotic spindles. This was associated with an important augmentation of the cytotoxic effect of etoposide. In contrast, a similar temperature shift of Phe-132 cells, which express mutant p53, had no effect on either immunostaining with MPM-2 or the cytotoxicity. Taken together, our results indicate that wild-type p53 can override the etoposide-induced G2 block in at least some cell types. These data propose a new role for p53 in the cell death induced by chemotherapeutic agents and may have important implications for gene therapy.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.