Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

Effect of aphidicolin on Friend erythroleukemia cell maturation

Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha, was examined as a potential tool to evaluate the relationship between proliferative and differentiative events in Friend erythroleukemia cell (FELC) maturation. Since FELC can be induced to differentiate along the erythrocytic pathway with a variety of inducing agents, the effects of aphidicolin were tested on proliferating FELC and cells which were induced to differentiate with the potent inducer, hexamethylene bisacetamide (HMBA). Exposure of FELC to aphidicolin resulted in unbalanced growth within 24 h, as reflected by abnormally large cells, compared with untreated cells. In the presence of 10 or 50 microM aphidicolin, 75-90% of cells became differentiated (benzidine+ cells) within 48 h, although by 72 h cells treated with aphidicolin were non-viable as determined by trypan blue staining. A wider range of aphidicolin concentrations was tested in an effort to determine the optimal concentration of aphidicolin that maximally induced differentiation with minimal loss of cell viability. Continuous exposure of FELC from 24-96 h with doses of aphidicolin ranging from 0.5 to 50 microM was more effective for differentiation induction than was short-term exposure (1, 2, 4, 12 h) to the drug, although 1 h of exposure significantly (p less than 0.01) increased differentiation (28.1 +/- 7.8%) compared with untreated cells (2.7 +/- 1.0%). When cells were treated with HMBA (5 mM) and aphidicolin (1, 5, 10 microM), in combination, aphidicolin shifted the time of onset of differentiation from 72 to 48 h, but did not act synergistically or additively with HMBA; nor was the induction effect of aphidicolin changed by HMBA. In contrast, suboptimal doses of aphidicolin (0.5 microM) in combination with HMBA (2.5 mM) produced an additive effect on FELC differentiation. In addition, [3H]thymidine experiments demonstrated that aphidicolin reversibly blocked FELC in S phase and at G1-S interface of the cell cycle. These results indicate that aphidicolin can induce the differentiation of FELC, and that a complete round of replicative DNA synthesis is not required for differentiation to occur.     

Terminal differentiation of human erythroleukemia cell line K562 induced by aphidicolin

To analyze the relationship between differentiation and DNA replication, the effect of aphidicolin, a specific inhibitor for DNA polymerase alpha, was measured with respect to erythroid differentiation and activities of DNA polymerases alpha, beta, and gamma. Five micromolar aphidicolin completely blocked the growth of K562 cells and caused 80% of cells to become hemoglobin positive after 5 days exposure. The cessation of K562 cell growth induced by aphidicolin was irreversible, whereas the inhibition of HeLa cell growth was completely reversible. The enzyme activity of DNA polymerase alpha of K562 cells showed a 50-110% increase with aphidicolin treatment as compared to control K562 cells; activities of DNA polymerases beta and gamma were not affected. These features sharply contrasted with the erythroid induction of the same cells by hemin, where cell growth was not suppressed and DNA polymerase alpha was not increased but rather decreased. The enzyme activity of DNA polymerase alpha remained high even after removal of aphidicolin from the culture medium. These results suggest that treatment with aphidicolin might induce an accumulation of protein factors for replication and/or differentiation, causing rapid cell differentiation of cells without cell division.     

Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.

The effects of aphidicolin, a specific inhibitor of DNA polymerase α, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 × 10⁻⁶ M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.     

Response to auxin by cells ofRiella helicophylla during reversible arrest in different cell-cycle phases

When cells of the unistratose meristem ofRiella helicophylla (Bory et Mont.) Mont. are reversibly arrested at G1/S transition by treatment with the inhibitor of thymidylate synthase 5-fluorodeoxyuridine, with the inhibitor of DNA polymerase , aphidicolin, or with an inhibitor of late DNA synthesis, 5-aminouracil, they continue to expand. Simultaneous supply of auxin enhances cell expansion, while simultaneous addition of the auxin antagonistp-chlorophenoxyisobutyric acid prevents cell enlargement. When the meristematic cells are reversibly arrested during G1 phase by treatment with chlorsulfuron, an inhibitor of acetolactate synthase, cell size remains unchanged, but it increases when auxin is supplied simultaneously. Simultaneous application of chlorsulfuron during treatment with 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil, prevents cell expansion. After recovery from 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil treatment, the cellular pattern of the meristem is severely disturbed, while in combination withp-chlorophenoxyisobutyric acid or chlorsulfuron, meristem differentiation is almost unaffected. During reactivation of divisional functions in mature cells induced by isolation of tissue fragments, blockage of DNA synthesis by aphidicolin causes an augmentation of rhizoid initials which are characterized by enhanced RNA synthesis. Exogenous supply of auxin is required for outgrowth of these rhizoid initials, while, in untreated fragments, auxin for rhizoid growth is provided probably by the dividing cells. When reactivation of divisional functions in tissue fragments is reversibly inhibited by chlorsulfuron, no changes in the cells are discernible and application of auxin has no effect; after release from blockage the cells regenerate like those in untreated fragments. The results suggest that the phases of the cell cycle differ with regard to auxin synthesis and competence to respond to auxin. Probably, during cycle inhibition at G1/S or S a rising auxin level causes disintegration of cell-cycle events.Abbreviations APH aphidicolin - 5-AU 5-aminouracil - CS chlorsulfuron - 5-FdUrd 5-fluorodeoxyuridine - PCIB p-chloro-phenoxyisobutyric acid Part of doctoral thesis, University of Kassel, GermanyI thank Professor Luise Stange (this Institute) for her suggestions and many stimulating discussions. This research was supported by a scholarship of the Otto-Braun-Fonds and by a grant of the Deutsche Forschungsgemeinschaft to Professor Stange.     

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.     

Aphidicolin induces alterations in Golgi Complex and disorganization of microtubules of HeLa cells upon long-term administration

Treatment of HeLa cells with aphidicolin at 5 or 0.5 μg/ml induced cell cycle arrest at G₁/S or G₂/M phase, respectively, and was accompanied by unbalanced cell growth. Long-term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti-Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh-like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin-arrested cells appeared partially broken up and seemed to have converted to a vesicle-like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 μg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organiz-ing center (MTOC). On the other hand, treatment with aphidicolin at 5 μg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 μg/ml aphidicolin when cycloheximide was added simultaneously to the culture. J. Cell. Physiol. 176:602–611, 1998. © 1998 Wiley-Liss, Inc.     

Nuclear and Organelle DNA Replication during Spore Germination in Bryophytes and Equisetum

In contrast to ferns, the spore nuclei of the moss, Funaria hygrometrica, are in the G₂ phase. They do not incorporate the thymidine analogue BUdr before the first mitosis. When spores germinate in the presence of aphidicolin, an inhibitor of the (nuclear) DNA polymerase alpha, they give rise to two-celled, negatively phototropic protonemata, consisting of relatively long and thin cells. Determinations of nuclear DNA content further confirm the presence of G₂-nuclei in Funaria spores. Spores of Pogonatum urnigerum and of Equisetum hyemale likewise contain G₂-nuclei, as shown by BUdr incorporation or experiments with aphidicolin, respectively. In germinating Funaria spores, the organelle DNA is synthesized very early and independent of nuclear DNA. A gradient in replication of organelle DNA is found only in the long tip cells which are formed in the presence of aphidicolin.     

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL‐18a cell line that stably expresses the fusion protein chIL‐18 was constructed and the enhanced green fluorescence protein connected through a (G₄S)₃ linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL‐18a cells (1 × 10⁵) were inoculated in 60‐mm culture dishes containing 5 mL of media to achieve 50%–60% confluence and were cultured in the presence of the cycle‐specific inhibitors 10058‐F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058‐F4, aphidicolin, and colchicine, respectively, the aphidicolin‐induced single cells showed higher fusion protein levels than did the 10058‐F4‐ or colchicine‐induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin‐treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN‐γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581–591, 2016     

Characterization of a Mutant of Toxoplasma gondii Resistant to Aphidicolin1

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.     

Synergistic effect of aphidicolin and ethanol on the induction of common fragile sites

Summary The effect of ethanol on the frequency of aphidicolin-induced common fragile sites was studied using lymphocyte cultures from two normal women. Aphidicolin was added to the cultures at a final concentration of 0.2 M and ethanol at 0.02%, 0.1%, 0.2%, 0.5%, and 1%, both during the last 26 h of culture. The frequency of common fragile sites increased from 296% in subject 1 and 201% in subject 2 with aphidicolin plus 0.02% ethanol, to 765% and 823%, respectively, with aphidicolin plus 1% ethanol. Ethanol alone added to cultures did not induce common fragile sites. The gaps and breaks induced by aphidicolin plus ethanol were highly nonrandom. Altogether, 35 common fragile sites were identified. The addition of 1% ethanol to aphidicolin increased both random and nonrandom gaps and breaks as compared with that of 0.02% ethanol. Dimethyl sulfoxide added to culture at final concentrations of 0.02% to 1% did not change the frequency of aphidicolin-induced fragile sites. The frequency of fluorodeoxyuridine-induced fragile sites was not affected by the addition of 0.02% to 1% ethanol. It was thus concluded that ethanol enhances the aphidicolin-induced fragile sites, possibly inhibiting the repair mechanism of gaps and breaks induced by aphidicolin.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors

Summary Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K _m for dCTP and the apparent K _i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.Abbreviations ara-A 9--d-arabinofuranosyl adenine - ara-C 1--d-arabinofuranosyl cytosine - aph aphidicolin     

Accumulation of Betacyanin in Phytolacca americana Cells and of Anthocyanin in Vitis sp. Cells in Relation to Cell Division in Suspension Cultures

In suspension cultures of Vitis sp., maximal accumulation ofanthocyanin was observed during the stationary phase. Accumulationof anthocyanin occurred in parallel with the cessation of celldivision under conditions such as a reduction of the concentrationof phosphate in the medium, or the presence of aphidicolin,an inhibitor of DNA synthesis. By contrast, in suspension culturesof Phytolacca americana, aphidicolin inhibited the accumulationof betacyanin and cell division. When aphidicolin was removedfrom cells by washing, partially synchronized division of cellswas induced and the accumulation of betacyanin also occurred,in conjunction with cell division. In the absence of phosphatefrom the medium, cell division did not occur and accumulationof betacyanin also ceased. Readdition of phosphate to cellsstarved for phosphate induced both cell division and the accumulationof betacyanin. These results indicate a positive correlationbetween the accumulation of betacyanin and cell division inPhytolacca which contrasts with a negative correlation betweenthe accumulation of anthocyanin and cell division in Vitis. (Received April 17, 1989; Accepted December 23, 1989)     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Nuclear-Cycle Dependence of the Development of the Preprophase Band in Tobacco BY-2 Cells

When tobacco BY-2 cells that had been treated with aphidicolinfor 24 h were cultured in the absence of aphidicolin, DNA synthesiswas initiated within 1 h. DNA synthesis was completed within4 h and then the preprophase band of microtubules (PPB) developed.However, when cells that had been treated with aphidicolin werecultured in the absence of aphidicolin for 1 h and then againin its presence, DNA synthesis, which was initiated during thehour in the absence of aphidicolin, was not completed withina further 10 h in the presence of aphidicolin. Moreover, thePPB did not develop even after the PPB had appeared and disappearedin cells that were cultured contemporaneously in the continuedabsence of aphidicolin. The development of the PPB seems to be causally associated withthe nuclear cycle of cell division in tobacco BY-2 cells. Thisprocess seems to require the completion of the replication ofall, or almost all, of the nuclear DNA. (Received July 25, 1992; Accepted November 24, 1992)     

Meiotic restriction in emmer wheat is controlled by one or more nuclear genes that continue to function in derived lines

Highly fertile F₁ hybrids were made between Triticum turgidum L. ssp. turgidum (2n = 28, AABB) and Aegilops tauschii Coss. (2n = 14, DD) without embryo rescue and hormone treatment. The F₁ plants had an average seed set of 25%. Approximately 96% of the F₂ seeds were able to germinate normally and about 67% of the F₂ plants were spontaneous amphidiploid (2n = 42, AABBDD). Cytological analysis of male gametogenesis of the F₁ plants showed that meiotic restitution is responsible for the high fertility. A mitosis-like meiosis led to meiotic restitution at either of the two meiotic divisions resulting in unreduced gametes. Test crosses of the T. t. turgidum–Ae. tauschii amphidiploid with Ae. variabilis and rye suggested that the mitosis-like meiosis is controlled by one or more nuclear genes that continue to function in derived lines. This discovery indicates a potential application of such genes in producing double haploids.     

Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.     

The effect of aphidicolin on the rate of DNA replication and unscheduled DNA synthesis of Bloom syndrome and normal fibroblasts

Summary Ultraviolet radiation induced more unscheduled DNA synthesis (UDS) in ten Bloom syndrome (BS) fibroblast strains than in control cells, but this difference could be suppressed by aphidicolin treatment in at least nine BS strains. Aphidicolin, 1 and 5 g/ml, were required to inhibit by 30% the UDS of BS and control cells respectively, but the DNA replication of BS cells did not prove abnormally sensitive to such an inhibitor. These findings are discussed in relation to current knowledge of the action of aphidicolin and hypotheses of the metabolic defect in BS.     

p53 mediates senescence-like arrest induced by chronic replicational stress

Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.