A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).     

Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation

The role of various segments (gag or v-abl) of the Abelson murine leukemia virus (A-MuLV) genome in both lymphoid cell and fibroblast transformation was examined by deletion of areas from cloned, plasmid DNA representations of the genome. The deleted plasmids were tested by transfection into fibroblasts and by infection of bone marrow cells using virus stocks derived from the fibroblast transfectants. Deletion of gag coding sequence from the A-MuLV protein did not affect fibroblast transforming activity but abolished lymphoid transforming activity. The gag- A-MuLV genomes were very unstable in transformed fibroblasts leading to large secondary deletions in v-abl sequences. The gag- A-MuLV proteins also had lower autophosphorylation than their gag+ counterparts although cells transformed by gag- virus had a normal elevation of protein-linked phosphotyrosine. Systematic deletion of v-abl sequences showed that only 45,000 to the 130,000 molecular weight of v-abl sequence in the A-MuLV protein is needed for fibroblast transformation and, at most, slightly more is needed for lymphoid cell transformation.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.     

Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.     

The induction of DNA synthesis in the chick red cell nucleus in heterokaryons during the first cell cycle after fusion with HeLa cells.

Induction of DNA synthesis in embryonic chick red cells has been examined during the first and second cell cycles after fusion with HeLa cells synchronized in different parts of G1 and S-phase. The data indicate that: (i) the younger the embryonic blood the more rapidly the red cells are induced into DNA synthesis; (ii) the greater the ratio of HeLa to chick nuclei in the heterokaryon, the more rapidly the induction occurs; (iii) DNA synthesis in the chick nucleus can continue after the HeLa nucleus has left S-phase and entered either G2 or mitosis; (iv) the induction potential of late S-phase HeLa is somewhat lower than that of early or mid S-phase cells; (v) less than 10% of the chick DNA is replicated during the first cycle after fusion and only a small proportion (15%) of the chick nuclei approach the 4C value of DNA during the second cycle after fusion; (vi) the newly synthesized DNA is associated either with the condensed regions of the nucleus or with the boundaries between condensed and non-condensed regions; (vii) the chick chromosomes at the first and second mitosis after fusion are in the form of PCC prematurely condensed chromosomes); they are never fully replicated and are often fragmentary; (viii) DNA synthesis in the chick nuclei is accompanied by an influx of protein (both G1 and S-phase protein) from the HeLa component of the heterokaryon.     

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.     

Sex chromatin and X chromosome replication patterns and incidence of sex chromatin in canine cell cultures

In order to provide evidence as to whether sex chromatin (SC) of interphase cells is equivalent to the late replicating X chromosome in female mammalian cells, time-lapse cinephotometric and autoradiographic methods were used to give precise data for comparison of the DNA replication patterns of SC with that of each of the X chromosomes throughout the S period. Canine kidney epithelial cells were selected because they have distinct large metacentric X chromosomes and typical SC. Time-lapse cinephotometry was used to avoid possible alteration of DNA synthesis by chemical cell synchronization agents. Determination of the incidence of SC during the stages of the cell life cycle of proliferating cells of the same origin was performed in order hopefully to clarify conflicting reports on the subject. Our results clearly show that time and intensity of the SC replication throughout S period is like that of the late replicating X chromosome and unlike that of the early replicating X chromosome. The incidence of SC in proliferating cells in culture was found to vary with the stage of the cell life cycle, increasing with increasing postmitotic interval — least in G1, greater in S, and greatest in G2. The SC incidence increased strikingly from G1 to S and a less marked increase was observed between S and G2.     

DNA-synthesis in synchronized L-cells after irradiation during the G1-phase of the cell cycle

Mouse fibroblast L-929 cells synchonized by mitotic selection were irradiated during the G1-phase of the cell cycle with a dose of 1000 rad. The rat of DNA synthesis was measured by 3H-thymidine incorporation, and the progression of the cells through the cell cycle was determined using a pulse-cytophotometer. Irradiation caused a decrease in the rate of DNA synthesis to half the control value, and an extension of the S-phase to twice its normal duration.     

Intracellular distribution of DNA methyltransferase during the cell cycle

The intracellular distribution of DNA methyltransferase has been analyzed in synchronously proliferating human cells. The localization of DNA methyltransferase was determined immunocytochemically using monoclonal antibodies directed against this enzyme. DNA methyltransferase was found to accumulate predominantly in nuclei with weak cytoplasmic staining. The DNA methyltransferase antigen was absent in early G1 phase, appeared in late G1 prior to the onset of DNA synthesis and persisted throughout S and G2 phases of the cell cycle. Mitotic cells showed a particularly strong staining intensity. These results show that DNA methyltransferase levels fluctuate during the cell cycle. This has possible implications on the stability of the DNA methylation pattern.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1–3 μg glycolipid mR595/10⁵ cells and those of the low order fix about 6 μg glycolipid mR595/10⁶ cells. Ca⁺⁺ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37°C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only β-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.     

Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.     

Sequence of activation of template biosynthesis in normal and transformed human cells after synchronization by a double thymidine block

The sequence of matrix biosyntheses of DNA, RNA and various proteins in normal and transformed human fibroblasts in the first mitotic cycle after synchronization of cells by double thymidine block was studied. Two important regularities of synthesis of acid-soluble histone-like and acid-insoluble proteins in normal and transformed cells were established. In normal fibroblasts, the synthesis of both acid-soluble and acid-insoluble proteins is minimal before DNA replication and maximal in the G2-phase; that in transformed cells is maximal after removal of the thymidine block and decreased in the G2-phase. In normal fibroblasts, the synthesis of acid-insoluble proteins is maximal before, while that of acid-soluble ones--after the maximum of DNA synthesis. In transformed cells the situation is opposite. RNA synthesis in normal and transformed cells is stimulated at the end of the G2-phase. In normal cells, protein synthesis is coupled with the activation of RNA synthesis, whereas in transformed fibroblasts protein synthesis occurs, in all probability, in the next mitotic cycle. These differences are especially well-pronounced in the expression of some LMG proteins. It is concluded that in transformed cells the regulatory control over the coupling of matrix biosyntheses is impaired.     

An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells.

Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in G0-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in G0-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.     

Apoptotic cell death in rat embryo fibroblasts transformed by EiA + cHa-RAS oncogenes after gamma irradiation

A mechanism of apoptotic death of normal rat embryo fibroblasts and of those transformed by E1A + cHa-Ras oncogenes following gamma irradiation has been investigated. The E1A + cHa-Ras transformed cells were shown to express wild type p53 which was able to trans-activate a reporter pG13-luc Plasmid. As a result of trans-activation, an accumulation of universal inhibitor of cyclin-dependent kinases--p21/Waf1 protein and an increase in the proportion of p21/Waf1 expressing cells were observed, The accumulated p21/Waf1 was found to bind with PCNA. The association with PCNA, however, did not lead to suppression of DNA replication according to the data of iododeoxyuridine (IdUr) incorporation. A high proportion of S-phase cells, in combination with cell cycle blocking in G2-phase, promoted polyploidization of E1A + cHa-Ras transformed cells after gamma irradiation. The polyploidic cells with DNA content equal and higher than 8c die 48-72 h following irradiation due to apoptosis. A significant proportion of E1A + cHa-Ras cells with incorporated IdUr contains labeled micronuclei, the fact being a morphological evidence of apoptosis of cells in S-phase of the cell cycle.     

Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.