Hydroxyurea does not prevent synchronized G1 chinese hamster cells from entering the DNA synthetic period

Walters, R.A.; Tobey, Robert A.; Hildebrand, C.E.

doi:10.1016/s0006-291x(76)80294-x

Cited by 86 publications

(36 citation statements)

References 15 publications

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“…Hydroxyurea prevents progression of exponentially growing cells traversing S phase and either arrests other cells at the G1-S boundary or allows these cells to slowly move into S phase (22). (2'-5')An polymerase activity did not increase in cells treated for 24 h with hydroxyurea (Table 2).…”

Section: Resltltsmentioning

confidence: 97%

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

Krishnan¹,

Baglioni²

1981

Mol. Cell. Biol.

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(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

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Section: Resltltsmentioning

confidence: 97%

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

Krishnan¹,

Baglioni²

1981

Mol. Cell. Biol.

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“…Not only is the drug capable of completely preventing replication fork progression for extended intervals (11), but we were unable to isolate cell lines displaying more than 5-10-fold higher resistance to mimosine than the starting cell line, and then only after a selection regimen lasting more than 18 months (20). 13 Although there are a few older reports describing mimosine's effects on tumors in rat models (49,50), perhaps this interesting drug should be revisited in light of its possible effects on SHMT.…”

Section: Figmentioning

confidence: 99%

“…12). After the 2.5-h lag, mimosine effectively prevents S phase cells from progressing any further in the cell cycle for at least 48 h, as assessed by fluorescence-activated cell sorter analysis; this contrasts with aphidicolin and hydroxyurea, which are relatively leaky even at high concentrations and allow cells to slowly traverse the S period (11,13). When added to cells as they attempt to cross the G 1 /S boundary, mimosine completely prevents the formation of replication forks in the dihydrofolate reductase origin in CHO cells, while aphidicolin and hydroxyurea do not (12,14).…”

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confidence: 99%

Mimosine Targets Serine Hydroxymethyltransferase

Lin

Falchetto

Mosca

et al. 1996

Journal of Biological Chemistry

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The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G 1 /S boundary, and blocks DNA replication when added to S phase cells after a lag of ϳ2.5 h. We have shown previously that [ 3 H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [ 3 H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly ؊ cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly ؊ cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.Our laboratory's interest is the regulation of DNA synthesis in mammalian cells and, in particular, the nature of origins of replication. Although it is known that mammalian DNA is replicated from bidirectional origins spaced ϳ100 kilobase pairs apart (1), the molecular mechanisms of this process remain elusive (see Ref. 2, for review).In the absence of a viable assay for identifying the genetic elements (replicators) that control initiation in mammalian cells, attention has been focussed on determining the positions at which replication initiates, which should lie close to replicators. This approach requires methods for obtaining cell populations in which initiation at a given origin is occurring at the same time. In a commonly used synchronization protocol, cells are first arrested in the G 0 (non-proliferating) compartment by nutritional or serum starvation, followed by release into an inhibitor of DNA synthesis (e.g. Refs. 3-5). The drug treatment is enforced for a time long enough to allow all cells in the population to arrive at the beginning of the S period (a time when at least some origins are sure to be firing); the drug is then removed, allowing cells to enter S in a semi-synchronous wave. Unfortunately, this protocol is not entirely satisfactory for examining initiation events at the beginning of S, because even the most efficacious replication inhibitors do not inhibit initiation per se; rather, they slow the rate of replication fork movement by affecting DNA polymerases (e.g. aphidicolin (6)) or by lowering deoxyribonucleotide pools (e.g. hy...

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“…On the other hand, the difference in the response of replicative versus repair synthesis to HU is not obvious; the substrate affinities of the known DNA polymerases do not vary enough to provide a satisfactory rationale (4,6). Moreover, the proposed mechanism cannot explain why HU-blocked cells do not stop at the Gl/S boundary but start replication and accumulate in the early S phase (10,36).…”

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confidence: 92%

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Wawra

Wintersberger

1983

Mol. Cell. Biol.

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Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the Gl/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.Hydroxyurea (HU) is a specific inhibitor of DNA replication (34). It suppresses the polymerization of new strands almost completely and causes the accumulation of short DNA fragments that cannot be further elongated (16,21,24,25,29). Repair synthesis is much less affected by the drug (34). It has generally been assumed that the block of the conversion of ribonucleotides to deoxyribonucleotides via the inhibition of the enzyme ribonucleoside diphosphate reductase is responsible for these effects of HU (17,18,20). On the other hand, the difference in the response of replicative versus repair synthesis to HU is not obvious; the substrate affinities of the known DNA polymerases do not vary enough to provide a satisfactory rationale (4, 6). Moreover, the proposed mechanism cannot explain why HU-blocked cells do not stop at the Gl/S boundary but start replication and accumulate in the early S phase (10, 36).The observation that, in a cell-free DNAsynthesizing system obtained from HU-treated cells, the inhibition was still partly effective although all triphosphates were present in theincubation mixture led us to reconsider the effect of HU. This phenomenon was specific for HU and could not be observed with fluorodeoxyuridine (FUdR), another substance which interferes with the precursor pool. Hence, it is unlikely that the observed lasting inhibition is due to the interference of HU with the biosynthesis of precursors. A more detailed investigation of this effect of HU is the subject of this report. (41) and grown in plastic petri dishes at 37°C in a water-saturated atmosphere with 7.5% C02, using DME medium containing 10% calf serum. After reaching confluence, cells stopped division, and they were usually us...

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Hydroxyurea does not prevent synchronized G1 chinese hamster cells from entering the DNA synthetic period

Cited by 86 publications

References 15 publications

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

Mimosine Targets Serine Hydroxymethyltransferase

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

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