Lawrence R. Gurley

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.

The metabolism of histone fractions: I. Synthesis of histone fractions during the life cycle of mammalian cells

Abstract The histone fractions f1, f2a1, f2a2, f2b, and f3 have been isolated from suspension tissue cultures of Chinese hamster cells. Amino acid analysis and polyacrylamide gel electrophoresis demonstrated that these Chinese hamster histone fractions are similar to calf thymus histone fractions obtained by the same method. Using the doublethymidine blockade method, Chinese hamster cultures were synchronized in their life cycle. The synthesis of all five histone fractions was clearly demonstrated to occur mainly in the S phase, each histone fraction being synthesized concomitantly with the other four fractions and accumulating in chromatin concomitantly with the increase in mass of DNA. A significant synthesis of all five histone fractions was also observed when DNA synthesis was inhibited in synchronized cultures by thymidine, suggesting the possibility that histones turn over in chromatin during thymidine block.

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.

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.

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase mu Bondapak C18 column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [3H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.

The metabolism of histone fractions. II. Conservation and turnover of histone fractions in mammalian cells.

Abstract During exponential growth of Chinese hamster cells in suspension culture, histone f1 was observed to turn over with a half-life of 74 hr, while histones f2a1, f2a2, f2b, and f3 did not turn over but were conserved for three cell generations. However, when synchronized cultures were blocked at the G 1 S boundary by inhibiting DNA synthesis with thymidine, histones f2a1, f2a2, f2b, and f3 were observed to turn over with halflives of 60, 58, 55, and 68 hr, respectively. This turnover indicated that prelabeled histones were being replaced by newly synthesized, nonlabeled histones under these conditions. Therefore, histone synthesis was not completely inhibited when net DNA synthesis was inhibited by thymidine. The turnover of histone f1 during thymidine block was more complex than the turnover of the other four histones. Early in thymidine block, when stable RNA synthesis was still occurring, f1 turned over with a half-life of 31 hr, but later in thymidine block, when stable RNA synthesis had stopped, the half-life was reduced to 47 hr (a rate somewhat similar to the turnover of the other histones during thymidine block). It appears that the turnover of f1 during exponential growth continued after thymidine block was applied and was added to the f1 turnover observed in late thymidine block, resulting in the rapid f1 turnover observed early in block. This rapid f1 turnover continued during block only as long as stable RNA synthesis continued, suggesting that the f1 turnover during exponential growth and part of the f1 turnover during early block may be functionally involved in some aspect of stable RNA synthesis requiring removal of f1 from the chromatin.

High-resolution disc electrophoresis of histones: I. An improved method☆

Abstract An improved method for high-resolution polyacrylamide gel disk electrophoresis of histones is presented. Electrophoretic patterns obtained by this technique for ribonuclease, trypsin, and a whole calf thymus histone preparation are displayed. The resolving power of this method is excellent and its simplicity, sensitivity, and reproducibility recommend it for routine analytical use.

The metabolism of histone fractions: III. Synthesis and turnover of histone f1

Abstract This report confirms and extends our previous measurements of histone f1 turnover in the chromatin of exponentially growing Chinese hamster cells. Acidic protein contaminants were removed from f1 preparations, and the resulting purified f1 was characterized as very lysine-rich histone. This purified f1 had a turnover half-life of 52.7 hr, an even greater turnover rate than that of whole f1. In cultures whose DNA replication was inhibited by thymidine, additional treatment with the aminonucleoside of puromycin or with actinomycin D failed to inhibit f1 turnover, thus indicating that the mechanism of f1 turnover is not dependent on continuing synthesis of a stable ribosomal RNA or an unstable messenger RNA. Analysis of f1 synthesis in the S phase of thymidine-synchronized cultures indicated that, although histone f1 is laid down in the chromatin complex concomitantly with DNA replication, part of this histone was synthesized at least 1 hr prior to its deposition in the chromatin. These data suggest that cells have the capacity to form a nonchromatin pool of histone f1.

Characterization of cDNA sequences corresponding to three distinct HMG-1 mRNA species in line CHO Chinese hamster cells and cell cycle expression of the HMG-1 gene

We have isolated cDNA clones encoding the high mobility group (HMG) protein HMG-1 in line CHO Chinese hamster cells. The cDNA clones correspond to the three HMG-1 mRNA species detected on Northern blots. Three different polyadenylation sites are found to be used. The three mRNA species of sizes 1.05, 1.45 and 2.45 kb are generated by differential polyadenylation at sites 115 nucleotides, 513 nucleotides and 1515 nucleotides downstream from the stop codon. A perfectly conserved putative poly(A) signal AAUAAA is present upstream of only one of the three poly(A) sites. Two homologous but imperfect sequences exist upstream from the other two poly(A) sites. All three HMG-1 mRNA species maintain significant levels throughout the M, G1 and S phases of the cell cycle and the rate of large HMG protein (HMG-1 and HMG-2) synthesis increases approximately two-fold from G1 to S phase.