Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

Background: Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. Methods: Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. Results: Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined ( or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. Conclusion: Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.     

Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition

Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.     

Flow cytometric detection of transforming growth factor-beta expression in rabbit articular chondrocytes (RAC) in culture--association with S-phase traverse.

We have previously shown that TGF-beta 1 decreased the entry of G0/G1-synchronized rabbit articular chondrocytes (RAC) into S-phase, whereas it enhanced the proliferation rate of actively dividing cells (asynchronous or S-phase-synchronized cells). The growth proliferative effect was accompanied by both increased DNA replication rate and G2/M delay. Since TGF-beta mRNA has been detected in chondrocytes, it was of interest to study the expression of the factor in correlation with the cell cycle of RAC. Using cytofluorometric analysis of both DNA content and TGF-beta protein level, we demonstrated that S-phase-synchronized RAC constitutively expressed TGF-beta, whereas G0/G1-synchronized cells only display very low levels of the factor. The data showed that the expression of TGF-beta is correlated with S-phase traverse since it increases with the percentage of cells in S-phase (less than 27% in G0/G1 to 70% in S-phase-synchronized cells). Moreover, exposure of RAC to TGF-beta 1 (1 ng/ml) for 24 h increased the percentage of positive cells, independently of the number of cells in S-phase, indicating that the factor may up-regulate its own expression. All together, these data suggest that TGF-beta could play a role in initiating the proliferation of articular chondrocytes during the early events of osteoarthritis and might take a part in the repair of cartilage matrix.     

Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment

The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.     

Paradoxical effects of iodine-125 decays in parent and daughter DNA: a new target model for radiation damage

Chinese hamster ovary cells were synchronized at the G(1)/S-phase boundary of the cell cycle and were pulse-labeled with (125)I-iododeoxyuridine 30 min after they entered the S phase. Cell samples were harvested and frozen for accumulation of (125)I decays during the first and second G(2) phase after labeling. Cell aliquots that had accumulated the desired number of decays were thawed and plated for evaluation micronucleus formation and cell death. Cells subjected to (125)I decays during the first G(2) phase after labeling exhibited single-hit kinetics of cell killing (n = 1, D(0) 41 decays/cell). In contrast, decays accumulated during the second G(2) phase killed cells with dual-hit kinetics (n = 1.9, D(0) 81 decays/cell). A similar divergence in the action of (125)I was noted for micronucleus formation. These findings indicate that the effects of (125)I varied depending on whether the decays occurred in daughter DNA (first G(2) phase) or parent DNA (second G(2) phase). Control studies with external X rays showed no such divergence of the action of radiation. To account for this paradox, a model is proposed that invokes higher-order chromatin structures as radiation targets. This model implies differential spatial arrangements for parent and daughter DNA in the genome, with DNA strands organized such that a single (125)I decay originating in daughter DNA damages two targets during the first G(2) phase, but identical decays occurring during the second G(2) phase damage only one of the targets.     

Serum C-19 steroid sulphates in females with clinical hyperandrogenism

Serum sulphates of 5-androstene-3 beta, 17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as unconjugated androstenedione (AD), testosterone (T) and 17 alpha-hydroxyprogesterone (17OHP), sex hormone binding globulin (SHBG) and the free androgen index (FAI) were measured by specific radioimmunoassay in girls with premature adrenarche (n = 9-16), and in hirsute women with (1) late onset 21 hydroxylase deficiency (n = 14), (2) polycystic ovarian disease (n = 28) and (3) idiopathic hirsutism (n = 74). Levels were also determined in females with androgenic alopecia (n = 35-45), in normal prepubertal girls (n = 9-14) and in normal adult women (n = 50-73). Mean serum concentrations of 5-ADIOL-S, 3 alpha-DIOL-S, DHEA-S, AD, T, and FAI were elevated and SHBG depressed, in all patient groups compared with controls, except for DHEA-S and T in patients with alopecia. We conclude that in addition to DHEA-S, 5-ADIOL-S may have a role as a pro-hormone in the synthesis of more potent androgens (T, DHT) in peripheral tissues such as skin; in addition, 3 alpha-DIOL-S may be a marker of peripheral androgen metabolism.     

Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine

Cell cycle kinetics after X-irradiation were studied in a solid rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine (BrdUrd) in cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S-phase cells in the tumor, only a fraction of both types of cells became labeled with BrdUrd during S-phase, even 24 h after injection. The diploid BrdUrd-labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid G1-phase cells could be observed. Only 25% of the tetraploid S-phase cells could be labeled by a single injection of BrdUrd (160 mg/kg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a "Mini Osmotic Pump" containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S-phase and 70% of cells in G2-phase were labeled. The fraction of labeled G2-phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation due to a radiation-induced G2 block in cycling tetraploid tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impulse cytophotometry studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 2. Lymphatic cells in blood

The DNA-content of mononuclear cells of the peripheral blood of infantile and juvenile ALL patients was investigated using Pulse Cytophotometry. The fraction of cells in S- and G2 + M-phase is significantly increased in comparison with samples of healthy probands. The fraction of DNA-synthesising cells (S-phase) of both peripheral blood (mononuclear cells) and bone marrow of leukemia patients cannot be significantly distinguished by mathematical methods. On the other hand, the fraction of cells in later phases of cell cycle (G2 + M-phase) is significant enhanced in the bone marrow in comparison with the peripheral blood. A high correlation was found between the number of leukocytes and fraction of G2 + M-phase cells in the peripheral blood of SR- and MR-patients. No correlation was found between the number of leukocytes and S-phase-fraction. The occurrence of aneuploid cell populations in the mononuclear fraction of peripheral blood in the acute state of ALL could be of importance for prognosis and regime of therapy.     

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.     

Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.     

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

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibroblast 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/10(7) cells during G1 to a high of 81.6 nmol/10(7) cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10(7) cells during late S-phase, followed by a second peak of 65.8 nmol/10(7) 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 deoxyribonucldoside triphosphates during G1 (0.3-1.3 pmol/10(7) cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5-6.4 pmol/10(7) cells). Declining levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7-10.7 pmol/10(7) cells) during G2-M.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.     

Mechanisms of 2''-deoxyguanosine toxicity in mouse T-lymphoma cells with purine nucleoside phosphorylase deficiency and resistance to inhibition of ribonucleotide reductase by dGTP.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and dGTP, resulting in feedback inhibition of ribonucleotide reductase (RR; EC 1.17.4.1) and hence DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of dGTP in G1-phase, but not in S-phase cells, because of the utilization of dGTP for DNA synthesis and limited capacity to synthesize dGTP from dG. These results support the hypothesis that dG/dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is RNA synthesis, which was inhibited at an early stage in dGuo-L cells.     

Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study

Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) = 1, and a duration of S phase and G2 + M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.     

Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells.

BrdU-Hoechst flow cytometry was employed to study the proliferation kinetics of blood lymphocytes from patients with Fanconi anemia (FA). Compared to controls, untreated FA lymphocytes show normal response to PHA stimulation, normal G0/G1 exit rates, and normal first S-phase durations. The G2 phase of the first cell cycle, however, is severely prolonged, and 24% of the recruited population become arrested during the first chromosome cycle (S, G2/M phases). The delay suffered during G2 appears to be compensated in part by a subsequent G1 phase duration that is unusually short for postnatal human cells (3.7 +/- 0.5 hrs). In analogy to what has been observed in other cell systems after experimental delays of the chromosome cycle, we therefore postulate that at least some FA cells enter their second growth phase without prior completion of the delayed chromosome cycle. Renewed replication would ensue in such cells without prior passing through mitosis and cytokinesis, leading to endoreduplication, which is a frequent finding in the FA syndrome.     

Functional significance of the serum/saliva testosterone ratio in various diseases]

Serum/salivary testosterone ratio (ST/SlvT) expresses the relationship in absolute values between bound and unbound testosterone. This ST/SlvT ratio in supposedly healthy men (n = 25) and women (n = 72) and in patients with several disorders, prostatic carcinoma (n = 19), varicocele (n = 9) and hirsute women (n = 16), has been studied. Both serum and salivary testosterone were measured by an RIA method. ST/SlvT ratio values found in healthy men (78.4 +/- 30.9) did not differ significantly from values found in the varicocele group (111.1 +/- 49.3), but a significant difference (p less than 0.001) from those found in men with prostatic carcinoma (12.3 +/- 7.2) was observed. When the ST/SlvT ratio values obtained in healthy women (18.1 +/- 7.3) were compared with those obtained in hirsute women (1.56 +/- 5.7) no significant differences were observed. The results obtained may indicate the dissociation among the hormone transport, testosterone metabolic clearance and hormone secretion by the salivary glands.     

Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2-kinase in mouse T-lymphoma cells

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.