DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids. — Reactivation of CE nuclei is associated with a loss of the tissuespecific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis.

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.     

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.     

Adult mouse kidneys become permissive to acute polyomavirus infection and reactivate persistent infections in response to cellular damage and regeneration.

Kidneys of newborn (but not adult) mice are normally high permissive for polyomavirus (Py) infection and readily establish persistent infections. We have proposed that ongoing cellular differentiation, which occurs in newborn mice, may be necessary for a high level of in vivo Py replication (R. Rochford, J. P. Moreno, M. L. Peake, and L. P. Villarreal, J. Virol. 66:3287-3297, 1992). This cellular differentiation requirement may also be necessary for the reactivation of a persistent Py kidney infection and could provide an alternative to the accepted view that reactivation results from immunosuppression. To examine this proposal, the ability of adult BALB/c mouse kidneys to support primary acute Py infection or to reactivate previously established persistent Py infections after kidney-specific damage was investigated. Kidney damage was induced by both chemical (glycerol, cisplatin, or methotrexate) and mechanical (through renal artery clamping to produce unilateral renal ischemia) treatments. We also examined the effects of epidermal growth factor (EGF), which enhances the rate of kidney regeneration, on Py replication. Using histopathologic techniques, in situ hybridization for Py DNA, and immunofluorescence for Py VP1 production, we established that both chemical damage and damage through renal artery clamping of adult kidneys promoted high levels of primary Py replication in these normally nonpermissive cells. This damage also promoted the efficient reactivation of Py replication from persistently infected kidneys, in the absence of immunosuppression. EGF treatment significantly increased acute Py replication and also reactivation in damaged kidneys. These results support the view that ongoing cellular division and differentiation may be needed both for high levels of acute Py replication and for reactivation of persistent infections in vivo.     

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.     

DNA polymerase and simian virus 40 infection of resting monkey cells: induction of aphidicolin resistant alpha-polymerase.

Tightly confluent monkey cell lines BSC-1 and CV-1 held in stale medium for several days exhibited an extremely low level of thymidine incorporation into cellular DNA. Yet, these cells contained a level of alpha-polymerase equal to about 15% of the level in rapidly dividing cells, and they still were capable of supporting replication of SV40 DNA. SV40 infection and culture in stale medium resulted in a four-fold induction of alpha-polymerase in CV-1 cells, whereas no change in alpha-polymerase level was observed in BSC-1 cells. Characterization of alpha-polymerase partially purified from infected CV-1 cells revealed that 80-90% of the enzyme activity was aphidicolin resistant. SV40 DNA replication in resting CV-1 cells, however, was aphidicolin sensitive. SV40 infection of resting CV-1 cells may induce an aphidicolin-resistant enzyme or lead to a modified alpha-polymerase species.     

Plk1 phosphorylation of Orc2 promotes DNA replication under conditions of stress

Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to maintain the functional pre-replicative complex (pre-RC) under DNA replication stress. Furthermore, the intra-S-phase checkpoint is activated in Orc2-S188A-expressing cells to cause delay of S-phase progress. Our study suggests a novel role of Plk1 in facilitating DNA replication under conditions of stress to maintain genomic integrity.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Studies on Differentiation without Cleavage in Chaetopterus: Effects of Inhibition of DNA Synthesis with Aphidicolin

The effects of aphidicolin – a powerful inhibitor of DNA polymerase α and of DNA replication – on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.     

Inhibition of DNA methylation is involved in transdifferentiation of myoblasts into smooth muscle cells

Despite the importance of cell fate decisions regulated by epigenetic programming, no experimental model has been available to study transdifferentiation from myoblasts to smooth muscle cells. In the present study, we show that myoblast cells can be induced to transdifferentiate into smooth muscle cells by modulating their epigenetic programming. The DNA methylation inhibitor, zebularine, induced the morphological transformation of C2C12 myoblasts into smooth muscle cells accompanied by de novo synthesis of smooth muscle markers such as smooth muscle alpha-actin and transgelin. Furthermore, an increase of p21 and decrease of cyclinD1 mRNA were observed following zebularine treatment, pointing to inhibition of cell cycle progression. This system may provide a useful model for studying the early stages of smooth muscle cell differentiation.     

Inhibition of myoblast differentiation by tumor necrosis factor alpha is mediated by c-Jun N-terminal kinase 1 and leukemia inhibitory factor

The proinflammatory cytokine, TNFalpha plays a major role in muscle wasting occurring in chronic diseases and muscular dystrophies. Among its other functions, TNFalpha perturbs muscle regeneration by preventing satellite cell differentiation. In the present study, the role of c-Jun N-terminal kinase (JNK), a mediator of TNFalpha, was investigated in differentiating myoblast cell lines. Addition of TNFalpha to C2 myoblasts induced immediate and delayed phases of JNK activity. The delayed phase is associated with myoblast proliferation. Inhibition of JNK activity prevented proliferation and restored differentiation to TNFalpha-treated myoblasts. Studies with cell lines expressing MyoD:ER chimera and lacking JNK1 or JNK2 genes indicate that JNK1 activity mediates the effects of TNFalpha on myoblast proliferation and differentiation. TNFalpha does not induce proliferation or inhibit differentiation of JNK1-null myoblasts. However, differentiation of JNK1-null myoblasts is inhibited when they are grown in conditioned medium derived from cell lines affected by TNFalpha. We investigated the induced synthesis of several candidate growth factors and cytokines following treatment with TNFalpha. Expression of IL-6 and leukemia inhibitory factor (LIF) was induced by TNFalpha in wild-type and JNK2-null myoblasts. However, LIF expression was not induced by TNFalpha in JNK1-null myoblasts. Addition of LIF to the growth medium of JNK1-null myoblasts prevented their differentiation. Moreover, LIF-neutralizing antibodies added to the medium of C2 myoblasts prevented inhibition of differentiation mediated by TNFalpha. Hence, TNFalpha promotes myoblast proliferation through JNK1 and prevents myoblast differentiation through JNK1-mediated secretion of LIF.     

Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Effect of aphidicolin on de novo DNA synthesis,DNA repair and cytotoxicity in γ-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase α and consequently of de novo DNA synthesis in human cells. We report here that in γ-irradiated normal human cells, aphidicolin (at 5 μg/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. γ-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase α is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA polymerase beta.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

Influence of external diffusible factors on myogenesis of the cells of the line L6

The influence of external diffusible factors on the terminal differentiation of cells of the myogenic line L₆ has been studied. The cultures were fed either with medium which had been depleted of mitogenic factors by previous incubation in the presence of myogenic cells, or with standard medium to which proteins secreted by myoblasts had been added. We present evidence that the length of the proliferative phase of the cultures is largely dependent upon environmental cues. However, by inhibiting DNA replication by a variety of means during this phase, we show that in order to differentiate, DNA synthesis is needed for myogenic cells of this line.Once the myoblasts have initiated their last presumptive round of DNA synthesis, they cannot be induced to undergo further DNA replication by environmental factors. Cloning experiments showed that, at this time, the cells lose their proliferative capacity. Our data strongly suggest that, at this stage, cells of line L₆ become irreversibly committed for differentiation. The fusion rate of the committed myoblasts could be significantly increased by proteins secreted by proliferating myogenic cells, but not by those secreted by myotubes.