DNA Synthesis Time Research Articles

Nuclear events during early development in gametophytes of Macrocystis pyrifera, and the temporal effects of a marine contaminant

The time course of DNA synthesis in developing haploid gametophytes of the giant kelp Macrocystis pyrifera was determined, and the effects of arsenic (As) on the temporally distinct nuclear events, DNA synthesis and subsequent nuclear division/translocation, were investigated to establish which of these specific events may be disrupted by this contaminant. Experiments were carried out on material collected from kelp beds near Santa Barbara, California from 1993–1994. Timing of DNA synthesis was determined during development by use of the fluorochrome, DAPI (4′,6-diamidino-2-phenylindole), and single-cell microspectrofluorometry. Zoospores, which result from meiosis, had already undergone two rounds of DNA synthesis at the time of release. The developing gametophytes underwent an additional two rounds of replication of DNA by 16 h of development, and following the first nuclear division/translocation, the gametophyte contained eight times the minimum DNA level throughout subsequent development. Both DNA synthesis and nuclear division/translocation, were found to be inhibited by As. Phosphate enrichment reduced the inhibitory effects of As on division/translocation of the nucleus, supporting the hypothesis that As interferes with phosphorylation. Gametophytes were more severely affected by As under light conditions, as opposed to dark, suggesting that photosynthesis may be more sensitive than dark metabolism.

Purification and characterization of bacteriophage T4 gene 59 protein. A DNA helicase assembly protein involved in DNA replication

The T4 bacteriophage gene 59 protein is required for normal T4 DNA replication. We have purified this protein to homogeneity in two steps and show that it binds both to single-stranded DNA and to the T4 gene 32 protein, a DNA single strand binding protein. In in vitro assays, covering DNA with 32 protein makes this DNA inaccessible to the 41 protein, the highly processive DNA helicase, that associates with the T4 DNA primase (gene 61 protein) to form an active primosome. However, the 59 protein brings about the rapid assembly of 41 protein onto single-stranded DNA, even if this DNA is covered with 32 protein. The 59 protein is therefore a DNA helicase assembly protein. The observed requirements for the 59 protein in the vivo T4 DNA replication are explained by there being two alternative pathways for loading the 41 protein onto a replication fork at early times of T4 DNA synthesis, with only a 59 protein-mediated pathway remaining operative for the recombination-mediated replication that dominates later in infection (Barry, J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 33063-33068).

Inhibition of tumorigenic potential and prostate‐specific antigen expression in LNCaP human prostate cancer cell line by 13‐cis‐retinoic acid

Prostate-specific antigen (PSA) is a member of the kallikrein family and has been an important biological marker for prostate cancer. The mechanisms regulating PSA expression in prostatic cancer cells are unclear. The present study was designed to elucidate the role of 13-cis-retinoic acid (RA) in regulation of PSA and the tumorigenic potential of the human prostate cancer cell line LNCaP. The growth regulation of LNCaP cells was examined by DNA synthesis and doubling time. The tumorigenic potential of prostate cancer cells was analyzed by soft agar colony-forming assay, in vitro invasion assay, type IV collagenase assay and binding to extracellular matrix assay. The nuclear receptors for retinoic acid (RAR alpha, -beta, -gamma and RXR alpha, -beta, -gamma) as well as PSA mRNA were determined by Northern blot using specific oligonucleotide probes. Our results suggest that 13-cis-RA significantly inhibits PSA secretion and expression both at the mRNA and protein levels compared with untreated cells. Electron microscopic studies suggest that after 13-cis-RA treatment, cells become more differentiated as they contain lumina, lined by plasma membrane and microvilli. Prostate cancer cell growth and tumorigenic potential after 13-cis-RA treatment was significantly decreased compared with controls. Nude mice tumorigenicity studies showed that 13-cis-RA-treated cells produced significantly smaller tumors compared with untreated cell tumors. There was also a significant increase in the expression of RXRa mRNA after 13-cis-RA treatment compared with untreated cells.

Hydroxyurea-Induced Synchrony of DNA Replication in the Kinetoplastida

We have developed a reliable and reproducible method to induce synchrony of the DNA synthetic cycle in the Kinetoplastida. The method involves treatment of cultures with 20 mMhydroxyurea (HU) and fetal bovine serum. Both stationary-phase and exponential-phase cultures can be synchronized. However, in the case of exponential-phase cultures the population doubling time and rate of DNA synthesis of the population influenced the time of exposure to HU. The treatment of kinetoplastids with 20 mM HU did not adversely affect the cells as judged by oxygen consumption, RNA, and protein content. We postulate that the requirement for high HU levels, which would be toxic to vertebrate cells, may be due to a lower affinity of kinetoplastid ribonucleotide reductase, the target enzyme for HU. Some of the kinetoplastids are pathogens of man and his food chain. Consequently, the development of a reliable technique for synchronization of the kinetoplastids should not only permit a detailed analysis of their cellular and molecular biology but provide a means to collect and characterize biochemical and immunochemical substances relevant to the infectious process.

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine‐labelled cells, using FCM‐derived data

Abstract. Chinese hamster ovary cells in vitro were pulse‐labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd‐labelled cells in relation to that of G1 and G2 cells, was calculated. RM was then used to calculate DNA synthesis time (TS), at all post‐labelling times (t). Since labelled cells in G2 and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G2 and M (Tg2+M) phases is finite, a non‐linear relationship exists between RM and post‐labelling time. Because of this, the use of a linear formula in the calculation of TS yields results that are affected by t. We found that RM data can be corrected with regard to TG2+M resulting in the derivation of a non‐linear TS formula. This non‐linear TS formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G1, S and G2+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd‐labelled cells in each window at every post‐labelling time. Plots of the fraction of BrdUrd‐labelled cells v. post‐labelling time were then made for each window. TS obtained in this way was in agreement with TS obtained with the corrected RM method. In conclusion, we present a method to calculate Ts which theoretically first makes the determination of RM independent of TG2+M, and secondly compensates for the non‐linear function of RM with post‐labelling time caused by accumulation of BrdUrd‐labelled cells in G2+ M.

CELL-CYCLE KINETICS OF PERMANENT GLIOMA-CELLS MEASURED BY PULSE LABELING WITH BROMODEOXYURIDINE

Cell phase distribution and cycle kinetics of six human glioblastoma cell lines were characterized after labelling with 5-Bromo-2-deoxyuridine (BrdUrd). Cycle time (T(c)), DNA synthesis time (T(s)), and potential doubling time (T(pot)) were compared with the actual doubling time (T(d)) of the growing cell population. Mathematical estimates closely correlated with T(d). Low labelling index (LI) correlated with short T(s) and vice versa. T(s) and LI allowed grouping of the cell lines in two clusters. The mean number of silver stained nucleolar organizer regions (mAgNORs) and percentage of cells with more than five AgNORs (pAgNOR) were counted. AgNORs closely related to LI. Low mAgNORs and pAgNORs correlated with fast T(s) among the clustered cell lines.

Abnormal DNA synthesis in polyamine deficient cells revealed by bromodeoxyuridine-flow cytometry technique.

Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis inhibitor 2-difluoromethylornithine (DFMO). At 1-4 days after seeding, the cells were labelled for 15-120 min with the thymidine analogue bromodeoxyuridine (BrdUrd) and they were then fixed directly after the labelling period. In addition, cells were labelled for 30 min and they were then allowed to progress in BrdUrd-free medium during a defined post-labelling time before fixation. An indirect immunofluorescence technique, using the monoclonal BrdUrd antibody and the intercalating stoichiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd-labelled cells to the mean DNA contents of G1 and G2 cells, a relative measure of the position of the BrdUrd-labelled cells was obtained (relative movement). Relative movement data, obtained from control and DFMO-treated cells fixed directly after BrdUrd labelling, indicated that DFMO-treated cells entered S phase at a normal rate, while their progression through S phase was impaired. DNA histograms of BrdUrd-labelled control cells fixed directly after labelling showed that most cells were found in early and late S phase, while DNA histograms of BrdUrd-labelled DFMO-treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. Analysis of relative movement of cells that were allowed to progress in BrdUrd-free medium after labelling showed that DFMO treatment resulted in a significant lengthening of the DNA synthesis time. Labelling index was significantly higher in DFMO-treated, growth-inhibited cells than in early plateau phase control cells indicating an S phase accumulation in the former cells.

Transient, meiosis-induced expression of the rec6 and rec12 genes of Schizosaccharomyces pombe.

Two meiotic recombination genes, rec6 and rec12, from Schizosaccharomyces pombe have been cloned by genetic complementation and their DNA sequences determined. Gene replacements demonstrated that the cloned fragments contained the rec6 and rec12 genes. Further analysis showed that the functional rec6 gene was within a 1.3-kb fragment and rec12 within a 1.7-kb fragment. The nucleotide sequences of these fragments revealed open reading frames (ORFs) predicting 143 amino acids for the rec6 gene product and 139 amino acids for the rec12 gene product. After pat1-114 temperature-induced meiosis, the transcripts of rec6 and rec12 were induced to maximal levels at 2-3 hr, at about the time of premeiotic DNA synthesis, but were present at much lower levels before and after this time. The transient induction of the transcripts and the phenotypes of the mutants suggest that the rec6 and rec12 gene products are involved primarily in the early steps of meiotic recombination in S. pombe. Near the rec6 gene are two additional ORFs potentially encoding proteins with homology to ribosomal protein S7 of Saccharomyces cerevisiae (ORF137) and to the homeodomain of developmental regulatory proteins (ORF201). The roles of these S. pombe ORFs remain to be determined.

Influence of interleukin-2 infusion on cell cycle kinetics in the Walker-256 carcinosarcoma

The effect of recombinant human interleukin-2 (IL-2) on tumor cell cycle kinetics was evaluated in rats bearing the Walker-256 carcinosarcoma. Seven days after implantation, tumor-bearing rats were infused intravenously with IL-2 at a dose of 500 mg/kg body weight or 5% dextrose for 6 h. Tumor cell mean DNA synthesis time (TDNA), labeling index, potential doubling time (Tpot), and growth fraction (GF) were determined in vivo by use of bromodeoxyuridine (BrdUrd) pulse labeling and bivariate BrdUrd/DNA analysis using flow cytometry. IL-2 treatment significantly reduced the relative number of S-phase cells by 11.9% and increased the number of cells in the G0/G1 phase by 9.4%. The labeling index was reduced from 41.3 +/- 2.5% to 32.7 +/- 1.2% (P < .01). Estimates of TDNA derived from the change in BrdUrd movement relative to DNA content were not affected by IL-2 treatment. Based on these cytokinetic changes, IL-2 infusion significantly increased tumor Tpot from 15.3 +/- 0.3 h to 21.5 +/- 0.2 h (P < .001) and reduced GF from 1.01 +/- 0.07 to 0.83 +/- 0.01 (P < 0.001). The inhibitory effect of IL-2 infusion on tumor cell growth, which may be either direct or secondarily mediated by other factors, contrasts with other stimulatory effects of this cytokine on lymphoid cell proliferation.

Proliferating cell nuclear antigen (PCNA) expression of megakaryocytopoiesis in normal human bone marrow and reactive lesions with special emphasis on HIV-myelopathy

A morphometric analysis was performed on bone marrow trephine biopsies using sequential double-immunostaining, to evaluate endoreduplicative activity of megakaryocytopoiesis. A total of 104 marrow specimens were studied with employment of monoclonal antibodies PC10 (anti-proliferating cell nuclear antigen-PCNA) and Y2/51-CD61 (anti-platelet glycoprotein IIIa). In addition to the control group patients included non-specific inflammatory changes, HIV-myelopathy with normal or decreased platelet counts, idiopathic thrombocytopenic purpura (ITP), and finally reactive thrombocytosis (TH). To exclude an undue overexpression of PCNA, in a comparative pilot study we also applied MIB1 (Ki-67 antigen) on normal bone marrow specimens. In accordance with the various modalities of cell-cycle marker expression, no significantly different findings were disclosed. PCNA-labelling index was relatively low, ranging from 0.8 to 1.7% of the total megakaryocytopoiesis (promegakaryoblasts to mature platelet-shedding megakaryocytes). A significant relationship between megakaryocyte size and PCNA-expression was determinable. This implies that some of the cases with a prevalence of small megakaryocytes, like ITP, have the tendency to show a higher proportion of positively-stained cells. Moreover, this feature confirms a hypothesis postulating a decrease in the time for DNA-synthesis (S-phase) and a relative prolongation of the G1/G2-phases of the cell-cycle at higher ploidy levels (large-sized megakaryocytes). On the other hand, it may be speculated that some of the hyperpolyploid giant megakaryocytes may have reached their endstage of endoreduplication and enter into G0-phase. In comparison with the control group and the other entities under study, a significant reduction of PCNA-reactivity was recognizable in HIV-myelopathy accompanied by thrombocytopenia.(ABSTRACT TRUNCATED AT 250 WORDS)

B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation

Small dense B cells are stimulated to proliferate by membranes prepared from activated helper T (Th) cell clones. In combination with Th2 lymphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th membrane requires the expression of CD40 ligand by the T cells, and initiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of the B cell response and found that Th membranes stimulated B cell proliferation and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of division rounds between day 2 and 5 of culture. IgM-secreting cells appeared in culture by day 3 and increased to reach a maximum, comprised of 30% of the viable cells, on day 5. IgG1-secreting cells appeared 12-24 h after IgM-secreting cells, and IgE-secreting cells did not appear until day 5. These data are consistent with a sequential model of isotype switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior to and during B cell proliferation for differentiation to Ig-secreting cells to proceed. This period corresponded closely to the time of maximum DNA synthesis. Consistent with sequential switching of Ig isotypes, differentiation to IgM secretion required the shortest exposure to lymphokines and IgE the longest. These experiments strongly suggest that the lymphokine-induced commitment to differentiate is made before DNA synthesis begins, although the lymphokine-delivered signals are necessary during DNA synthesis to support Ig class switching.

Dual modes of action of platelet-derived growth factor and its inhibition by trichostatin-A for DNA synthesis in primary cultured smooth muscle cells of rat aorta.

The effects of platelet-derived growth factor (PDGF) on the start and rate of DNA synthesis were investigated with or without (R)-trichostatin-A (TS-A) in primary cultured and synchronized smooth muscle cells (SMC) of rat aorta. After the SMC at the G0 phase were precultured with 10 and 100 ng/ml PDGF for 3 h and washed out,they were then stimulated with 3% fetal bovine serum (FBS). FBS-stimulated DNA synthesis was determined every 3 h for 24-30 h. The SMC pre-cultured with PDGF started DNA synthesis at an earlier time, dependent on the concentrations, suggesting an acceleration of competence. After the SMC were precultured with PDGF (30 ng/ml) plus TS-A (0.1, 0.3, 1.0 and 3.0 micrograms/ml) for 3 h and washed out, the SMC were then stimulated by 1% FBS. TS-A delayed the starting time of DNA synthesis in a concentration-dependent manner. During the prolonged culture with PDGF (1, 3, 10 and 100 ng/ml) and FBS (3%), the rate of DNA synthesis was more rapid than in cells pretreated with PDGF alone, suggesting an acceleration of progression. These results suggest that 1) PDGF stimulates DNA synthesis dually during the competence and progression phases, and 2) PDGF-induced competence is inhibited by TS-A in primary cultured SMC.

Effects of granulocyte-macrophage colony-stimulating factor and interleukin-3 on small cell lung cancer cells.

Nonhematopoietic malignant cells may express receptors for hematopoietic growth factors and respond to these peptides. The aim of the present study was to investigate whether small cell lung cancer (SCLC) cells may be stimulated to proliferate by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), which are currently used in clinical trials in combination with cytotoxic chemotherapy. We studied two SCLC cell lines, H69 and N417. The effects of GM-CSF and IL-3 were evaluated by studying clonal growth, 3H-thymidine incorporation, BUDR/DNA bivariate flow cytometry, c-myc and N-myc oncogene expression, and myeloid surface markers. Our experiments show that both GM-CSF and IL-3 can increase 3H-thymidine incorporation and cloning efficiency and reduce DNA synthesis time of H69 and, to a lesser extent, N417 cells, supporting the hypothesis that hematopoietic growth factors can stimulate the growth of some malignant nonhematopoietic cells in vitro. Further in vitro and in vivo studies are needed to determine whether clinical trials applying these factors for bone marrow recovery after chemotherapy of solid tumors may be hazardous by potentially stimulating growth of remaining tumor tissue.

Abrupt RNA changes precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro

We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.

Intra-tumoral heterogeneity of tumour potential doubling times (Tpot) in colorectal cancer.

Intra-tumoural heterogeneity of proliferation has been assessed by taking multiple biopsies from 30 colorectal cancers. Following in vivo IUDR labelling, dual parameter flow cytometry was used to measure tumour DNA index (DI) and labelling index (LI) and to derive DNA synthesis time (Ts) and potential doubling time (Tpot). Heterogeneity was seen for all parameters under investigation. Overall coefficients of variation (CV) and logarithmic transformation of Ts and Tpot (due to their non-gaussian distributions) indicate that LI (CV 25%) was the most variable parameter. Intra-tumoral heterogeneity in Tpot (lnTpot CV = 22%) was less than inter-individual variation (CV = 63%), suggesting that this variation should not be a limitation to the possible usefulness of this technique as an independent prognostic indicator. Correlations of Tpot values were examined between the shortest, the median and the value for a pooled homogenate sample from a single tumour. Using an homogenate, it was possible to accurately predict classification of tumour Tpot values as being below the median ('fast tumours') in 15 of 19 cases (79%). The data suggest that assaying an homogenate may allow a more rapid analysis of a multiply sampled tumour.

The competence progression model in CHO-K1 cells: The relationship between protein kinase C and immediate early gene expression in the insulin mitogenic signal

CHO-K1 cells grow in a defined medium with insulin, at physiological concentrations, as the only hormone. IGF-I can substitute for insulin. Quiescent cells require a 9–10-h lag, subsequent to the addition of insulin, to synthesize DNA. The phorbol ester, 12- O-tetradeconoylphorbol 13-acetate (TPA), cannot support growth of these cells, is a more effective inducer than insulin of c-fos, c-myc, c-jun, jun-B, Krox-20, Krox 24, fra-1 and JE, and induces fra-1, JE and c-myc with different kinetics from those of insulin. The addition of insulin + TPA to quiescent cells produces a synergistic effect on DNA synthesis but not on the expression of immediate early genes. Pretreatment of these cells with TPA or insulin decreases the required lag time for DNA synthesis by 3 h in a protein-synthesis-independent manner. These results, together with other experiments, demonstrate that [1] the insulin signal is independent of PKC, [2] insulin acts as a weak competence and a strong progression factor, while TPA behaves as a strong competence factor, and [3] the 9–10-h lag is made up of a 3-h period which is independent of protein synthesis, advancing the cells to a post-G o state of ‘competence’.

Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics.

In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colony-forming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (Ts), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34+ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a below-median LI (< or = 7.6%) and below-median Ts value (< or = 14.3 h). These trends were more pronounced in the group of de novo AML (n = 23), where the prolonged event-free survival in patients with below-median Ts reached statistical significance (p = 0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p = 0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.

Cell kinetics with in vivo bromodeoxyuridine assay, proliferating cell nuclear antigen expression, and flow cytometric analysis. Prognostic significance in acute nonlymphoblastic leukemia.

The proliferative characteristics of acute nonlymphoblastic leukemia (ANLL) were studied in vivo, and data were correlated with response to chemotherapy and survival. Sixty-five patients with untreated ANLL and 15 patients with solid tumors and normal bone marrow (BM) received 250 mg/m2 of bromodeoxyuridine (BUdR); bivariate flow cytometric (FCM) analysis then was used to measure cell BUdR incorporation and DNA content to obtain a complete set of kinetic parameters (i.e., BM BUdR-labeling index, DNA-synthesis time, potential doubling time [Tpot], and cell production rate). The percentage of blasts with positive results for proliferating cell nuclear antigen (PCNA) also was obtained by FCM analysis on the same BM samples, and these kinetic parameters were derived specifically for the ANLL proliferating compartment (growth fraction). Induction therapy, consisting of vincristine, arabinosylcytosine, and daunomycin, was administered subsequently to the patients with ANLL. Overall ANLL proliferative activity was lower than normal myelopoiesis, and a short Tpot was found to be a favorable factor for achieving complete remission (CR), the duration of CR, and survival. When the growth fraction was considered, however, ANLL proliferative activity was higher and more like that of normal BM. The kinetic differences detected in the PCNA-positive cells of patients with CR and no response and those with CR and survival durations above and below the median values for the entire series were highly significant in univariate analysis and retained a strong independent prognostic value when multivariate analysis was performed. These data show the clinical feasibility of a detailed study of cell kinetics by means of new FCM-based techniques and reinforce the clinical value of pretreatment proliferative activity in ANLL.

Role of Ca2+ in stimulation of DNA synthesis by epidermal growth factor and tumor promoters in cultured rat hepatocytes.

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 microM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 microM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca(2+)-containing media with Ca(2+)-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.

Effects of the Selective Protein Kinase C Inhibitor, Ro 31-7549, on the Proliferation of Cultured Mouse Epidermal Keratinocytes

We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1 microM], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 microM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50) = 4.4 microM]. The time course of [3H]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25 microM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 mM. This antiproliferative effect of TPA was largely reversed by 0.1 microM Ro 31-7549. In the proliferative culture system, 0.75 microM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.