Ronald A. Walters

Effects of Caffeine on Radiation-Induced Phenomena Associated with Cell-Cycle Traverse of Mammalian Cells

Caffeine induced a state of G(1) arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following X-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cells synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The X-ray-induced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeine-treated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed.

Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in chinese hamster cells

Abstract Variation of levels of the four deoxyribonucleoside triphosphate pools has been examined as synchronized Chinese hamster cells complete mitosis and traverse the cell cycle in preparation for division. The results indicate that; 1. 1. Mitotic cells have the largest pools of dATP, dGTP, and dTTP and that all four deoxyribonucleoside triphosphates are degraded as cells exit from mitosis. 2. 2. The pool of dCTP is maximal in late S-early G2, and degradation begins in G2. 3. 3. G1 cells have little or no deoxyribonucleoside triphosphates. 4. 4. The pool size of all four deoxyribonucleoside triphosphates increases just prior to initiation of DNA synthesis and increases throughout S. 5. 5. Cells treated with hydroxyurea in G1 accumulate dTTP, dCTP, and dGTP at the time that cells would normally have initiated DNA synthesis, but accumulation of dATP is completely inhibited as is initiation of DNA synthesis. 6. 6. The four deoxyribonucleoside triphosphates are not present in equimolar concentrations, pools in mid-S being 10 pmoles 10 6 cells , 27 pmoles 10 6 cells , 104 pmoles 10 6 cells , and 76 pmoles 10 6 cells for dGTP, dATP, dTTP, and dCTP, respectively. The pools of dGTP, dATP, dTTP, and dCTP are sufficient to support DNA synthesis for 1.0, 1.3, 5.2, and 3.8 min, respectively.

Evidence that x-irradiation inhibits DNA replicon initiation in Chinese hamster cells

Summary Analysis of DNA replicated 20–30 min after x-irradiation showed that the relative size distribution of single-strand DNA was deficient in DNA 7 daltons and enriched in DNA > 4 × 10 7 daltons. During this time, depressed thymidine incorporation into total DNA could be accounted for by failure to replicate DNA 7 daltons, with relatively little effect on larger DNAs. At 2 hr post-irradiation, synthesis of the entire size spectrum of DNA was depressed. By 4 hr post-irradiation, recovery of synthesis of all sizes of DNA was complete. The data indicate that x-irradiation preferentially inhibits replicon initiation, with relatively little effect on elongation.

Histone phosphorylation in late interphase and mitosis

Abstract Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the G 1 S boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G2-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G1, S, or G2-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.

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

Using very high concentrations of radioactively labeled thymidine, we show that synchronized G/sub 1/ cells treated with hydroxyurea entered the DNA synthetic period at a time and rate indistinguishable from that of untreated cells, although the rate of DNA synthesis was greatly reduced in the drug-treated cultures. The DNA synthesized in the presence of hydroxyurea was less than or equal to 1 x 10/sup 7/ daltons, all of which could be chased into bulk DNA of approximately 3.5 x 10/sup 8/ daltons within 3 hr after removal of hydroxyurea. Hydroxyurea synchronized cells are apparently not blocked at the G/sub 1//S boundary but in the S phase itself.

The metabolism of histone fractions. IV. Synthesis of histones during the G1-phase of the mammalian life cycle☆

Abstract The synthesis of histone fractions in the absence of DNA synthesis was measured using a new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G 1 -phase. It was found that such G 1 -arrested cells synthesize histones at a detectable but very low rate, about 2% of that of an S-phase cell. G 1 -cells traversing their life cycle (following synchronization by mitotic selection) were found to synthesize histones at 3.5 – 5.0% of an S-phase cell. Synthesis of histone in arrested G 1 -cells was accompanied by a turnover of histones on the chromatin. Histone f2a2 had the slowest turnover rate (187-hr half-life). Histone f1 had the fastest G 1 -phase turnover rate (97-hr half-life), which is only slightly less than the rate previously measured for f1 in exponential cultures. It is concluded that the naturally occurring turnover of histone f1 in these cells is not coupled to DNA synthesis. The high rate of histone synthesis in synchronized thymidine-blocked cells, compared to the very low rate of histone synthesis in G 1 -cells, indicates that synchronized thymidine-blocked cells are not biochemically G 1 -cells but are, rather, S-phase cells whose progression through S is arrested.

The metabolism of histone fractions: VI. Differences in the phosphorylation of histone fractions during the cell cycle☆

Abstract Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G 1 -phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G 1 -arrested and G 1 -traversing cells and active in the S-phase. The absence of f1 phosphorylation in G 1 -arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G 1 -traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G 1 -state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G 1 -traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G 1 -arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G 1 -traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity.

Rapid assembly of newly synthesized DNA into chromatin subunits prior to joining of small DNA replication intermediates

Abstract Nuclei from cells having the replicating DNA pulse-labeled with [3H]thymidine and the nonreplicating DNA uniformly labeled with [14C]thymidine were treated with micrococcal nuclease according to procedures which have been used to study the subunit structure of chromatin. Sedimentation analyses of chromatin from nuclease-treated nuclei, together with measurements of the size of newly synthesized DNA, indicate that (1) chromatin subunits near the replication fork are more susceptible to nuclease attack than subunits in non-replicating chromatin; (2) newly synthesized DNA is rapidly assembled into chromatin subunits prior to joining of small DNA replication intermediates; and (3) within 10 min after synthesis, DNA in newly replicated chromatin acquires a susceptibility to nuclease treatment similar to that of non-replicating chromatin.

Action of heparin on mammalian nuclei: I. Differential extraction of histone H1 and cooperative removal of histones from chromatin

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.

The metabolism of histone fractions. Phosphorylation and synthesis of histones in late G1-arrest.

Abstract The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G1 by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G1 slightly before the G 1 S boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G1, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G1, resulting in significant G1-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G1 before “new” f1 which is synthesized in S phase; and (c) G1-phase f1 phosphorylation does not require new histone or new DNA synthesis. Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G1-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G1-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G2 to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G1, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.