Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

Alphavirus M1 induces apoptosis of malignant glioma cells via downregulation and nucleolar translocation of p21WAF1/CIP1 protein

Alphavirus, a genus of arthropod-borne togavirus, is well-known for its pro-apoptotic capability. However, the underlying mechanism remains to be further clarified. Here, we have identified that M1, an alphavirus isolated in 1960s, targeted C6 malignant glioma cells for apoptosis. Flow cytometry analysis showed that more cells enter S-phase post M1 infection, and subsequently undergo a classic apoptosis. To elucidate the mechanism of S-phase arrest and its relationship to apoptosis, we tested the expression of several critical cell cycle regulatory proteins and found elevated phosphorylation of cyclin-dependent kinase 2 (CDK2), decreased expression of cyclin A and proliferating cell nuclear antigen (PCNA). Notably, the protein level of p21^WAF1/CIP1 was downregulated earliest and most effectively among all tested changes of cell cycle regulators, though its mRNA level was strongly upregulated. To evaluate the role of p21^WAF1/CIP1 in S-phase accumulation and subsequent apoptosis, we confirmed that exogenous p21^WAF1/CIP1 overexpression or treatment with roscovitine (a selective chemical inhibitor of CDK2) efficiently protected against apoptosis with a reduced S-phase accumulation Thus, it is indicated that the downregulation of p21^WAF1/CIP1 mediated C6 apoptosis via overactivation of CDK2. In addition, confocal microscopy showed that p21^WAF1/CIP1 totally translocated to nucleolus during M1-induced C6 apoptosis. Altogether, downregulation and nucleolar translocation of the p21^WAF1/CIP1 protein played an active role in M1-induced C6 apoptosis.     

Image analysis of in situ cell cycle related changes of PCNA and Ki-67 proliferating antigen expression

Abstract. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki-67 cell cycle related expression and distribution pattern analysed in the same cells. MCF-7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki-67. The major cell type was identified on each time sample as a function of the PCNA/Ki-67 pattern, and both antigens as well as DNA were quantified. During G₁ phase, the expression of PCNA greatly increased whereas Ki-67 content decreased. During S phase, nuclear Ki-67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G₂ phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki-67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G₁ cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G₁ cells had disappeared) tend to indicate that these cells have left the cycle by the end of G₁ phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki-67/PCNA content. The biggest fraction was PCNA negative and Ki-67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G₁. Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki-67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G₁ phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G₁. Some cells seem to go through early G₁ (G_1a and late G₁ (G_lb) while others may come out of mitosis committed to go through the following cycle by directly entering late G₁ compartment.     

Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21WAF1, Cdt1, cyclin A,PCNA and Ki-67 in relation to DNA replication in individual cells

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4^Cdt2 which regulates the licensing factor Cdt1 and p21^WAF1 during the G₁ to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21^WAF1, detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2′-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G₂ phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21^WAF1 and Cdt1 was seen at the end of G₁ phase and all DNA replicating cells were p21^WAF1 and Cdt1 negative. The loss of p21^WAF1 preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).     

The transient receptor potential vanilloid‐3 regulates hypoxia‐mediated pulmonary artery smooth muscle cells proliferation via PI3K/AKT signaling pathway

Objectves

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP channels family of Ca²⁺‐permeant cation channels. In this study, we aim to investigate the role of TRPV3 in pulmonary vascular remodeling and PASMCs proliferation under hypoxia.

Materials and methods

The expression of TRPV3 was evaluated in patients with pulmonary arterial hypertension (PAH) and hypoxic rats, using hematoxylin and eosin (H&E) and immunohistochemistry. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of TRPV3 on proliferation of PASMCs.

Results

We found that, in vivo, the expression of TRPV3 was increased in patients with PAH and hypoxic rats. Right ventricular hypertrophy measurements and pulmonary pathomorphology data show that the ratio of the heart weight/tibia length (HW/TL), the right ventricle/left ventricle plus septum (RV/LV+S) and the medial width of the pulmonary artery were increased in chronic hypoxic rats. Moreover, the expression of proliferating cell nuclear antigen (PCNA), Cyclin D, Cyclin E and Cyclin A, phospho‐CaMKII (p‐CaMKII) were induced by hypoxia. In vitro, we revealed that hypoxia promoted PASMCs viability, increased the expression of PCNA, Cyclin D, Cyclin E, Cyclin A p‐CaMKII, made more cells from G₀/G₁ phase to G₂/M + S phase, enhanced the microtubule formation, and increased [Ca²⁺]_i, which could be suppressed by Ruthenium Red, an inhibitor of TRPV3, and TRPV3 silencing has similar effects. Furthermore, the up‐regulated expression of PCNA, Cyclin D, Cyclin E and Cyclin A, the increased number of cells in G₂/M and S phase, and the enhanced activation and expression of PI3K and AKT proteins induced by hypoxia and in presence of carvacrol (an agonist of TRPV3), was significantly attenuated by incubation of LY 294002, a specific inhibitor for PI3K/AKT.

Conclusions

These findings suggest that TRPV3 is involved in hypoxia‐induced pulmonary vascular remodeling and promotes proliferation of PASMCs and the effect is, at least in part, mediated via the PI3K/AKT pathway.

     

Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G₂/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21^Cip1/WAF1, p27^kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action.     

Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

DNA polymerase α/primase (Polα) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Polα was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Polα and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Polα and by protein affinity chromatography of cell extracts derived from pure G₁-and S-phase cell populations on Polα affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Polα affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Polα (Copeland and Wang, 1991). With 5×10⁸ infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G₁-and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2×10⁹ non synchronous cells, 5×10⁸ G₁-phase cells were isolated. Chromatography of cell extracts derived from G₁-or S-phase cells on Polα affinity columns resulted in identifying several polypeptides in the range of 40–70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G₁-phase extracts.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

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

Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G₁-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G₁-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40^SIC1, very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G₁-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40^SIC1, restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.     

Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

DNA polymerase α/primase (Polα) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Polα was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Polα and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Polα and by protein affinity chromatography of cell extracts derived from pure G₁-and S-phase cell populations on Polα affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Polα affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Polα (Copeland and Wang, 1991). With 5×10⁸ infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G₁-and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2×10⁹ non synchronous cells, 5×10⁸ G₁-phase cells were isolated. Chromatography of cell extracts derived from G₁-or S-phase cells on Polα affinity columns resulted in identifying several polypeptides in the range of 40–70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G₁-phase extracts.     

Proliferating Cell Nuclear Antigen Bound to DNA Synthesis Sites: Phosphorylation and Association with Cyclin D1 and Cyclin A

Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G₁, or in G₂ + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G₁/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G₁/S phase boundary to drive activation of DNA replication factors.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

CRL4CDT2 Targets CHK1 for PCNA-Independent Destruction

CDT2 targets proteins involved in replication licensing (CDT1), cell cycle control (p21), and chromatin modification (SET8) for destruction by the CUL4-based E3 ligase (CRL4). CRL4^CDT2 recruits these substrates through interactions with chromatin-bound PCNA and ubiquitinates them exclusively on chromatin. Rereplication and G₂ cell cycle arrest are observed in CDT2-depleted cells. The rereplication phenotype has been attributed to an inability to destroy CDT1, but the molecular target important for G₂ cell cycle arrest in CDT2-depleted cells has not been identified. Here we identify CHK1 as a novel CRL4^CDT2 substrate and demonstrate that CHK1 activity is required for maintaining G₂ arrest in CDT2-depleted cells. We demonstrate that CRL4^CDT2 targets the activated form of CHK1 for destruction in the nucleoplasm rather than on chromatin and that this occurs in a PCNA-independent manner. Although both CRL1 and CRL4 ubiquitinate CHK1, we report that they bind CHK1 in distinct cellular compartments. Our study provides insight into how elevated CDT2 expression levels may provide tumors with a proliferative advantage.     

Hdm2- and proteasome-dependent turnover limits p21 accumulation during S phase

Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G₁ and occasionally G₂ phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G₁ and G₂ phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF^skp2 E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.     

Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized moused fibroblast cell line

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibrolast cells synchronized by isoleucine deprivation. Absolute pool sizes of ribonucleoside triphosphates were approximately 30 fold greater than those of the corresponding deoxyribonucleoside triphosphates. Of the ribonucleoside triphosphates, pool sizes of ATP exhibited the greatest change, increasing from a low of 32.7 nmol/10⁷ cells during G₁ to a high of 81.6 nmol/10⁷ cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10⁷ cells during late S-phase, followed by a second peak of 65.8 nmol/10⁷ with the onset of cell division. No significant changes in the pool sizes of UTP and GTP were found throughout the cell cycle. Of the deoxyribonucleoside triphosphates, pool sizes of pyrimidine deoxyribonucleoside triphosphates were approx. 5–10 fold greater than those of purine deoxyribonucleoside triphosphates. Low levels of deoxyribonucleoside triphosphates during G₁ (0.3–1.3 pmol/10⁷ cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5–6.4 pmol/10⁷ cells). Decling levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7–10.7 pmol/10⁷ cells) during G₂-M.