Robert A. Tobey

Cell-Cycle Analysis in 20 Minutes

Mithramycin added to mammalian cells fixed in aqueous ethanol is bound to DNA and fluoresces in direct proportion to the cellular DNA content. Quantitative fluorescence measurement by means of a high-speed flow system instrument provides a rapid method for cell-cycle analysis and for the first time permits continuous monitoring of cell-cycle kinetics during ongoing experiments.

Life Cycle Analysis of Mammalian Cells: III. The Inhibition of Division in Chinese Hamster Cells by Puromycin and Actinomycin

Analysis of the effects of actinomycin and puromycin on the G(2) and mitotic parts of the life cycle in Chinese hamster ovary cells grown in suspension and synchronized by thymidine treatment has been carried out. Rates of division of partially synchronized cell populations were measured in the presence and absence of the drugs, and various controls were performed to test for absence of complex side effects. Actinomycin produces a block 1.9 hr before completion of division, while puromycin produces a block almost coinciding with the initiation of mitosis. Evidence is presented that the puromycin block may be a double one, inhibiting one kind of protein synthesis that virtually coincides with the beginning of mitosis and another that occurs about 8 min earlier. The data are interpreted in terms of the time interval between messenger formation and its associated protein synthesis in this region of the life cycle. The various events studied have been provisionally mapped in the G(2) and mitotic periods of the life cycle.

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.

A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity.

Abstract The toxic trace metal Cd 2+ has been used to select a variant (designated Cd r ) of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd 2+ . Resistance of the Cd r cell to Cd 2+ -mediated cytotoxicity is not due to a decreased capability of the Cd r cell to accumulate Cd 2+ since Cd 2+ uptake in the Cd r cell is indistinguishable from that in the CHO cell at both toxic and subtoxic Cd 2+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd 2+ -binding proteins (metallothioneins) reveals that the Cd r cell has an increased capacity to induce metallothionein and to sequester intracellular Cd 2+ in metallothioneins. These results suggest that the greater competence of the Cd r cell to induce metallothionein is a major factor in the Cd 2+ -resistant phenotype of the variant.

Cell growth and division. IV. Determination of volume growth rate and division probability.

Volume growth rate and division probability functions for mammalian cells have been determined as functions of cell volume with good reproducibility and statistical precision using Coulter volume spectrometry and the equations of the Bell model. Results are compared with independent measurements on synchronous cultures. The slow rate of volume dispersion requires that the growth rate F(tau, V) be closely proportional to volume for cells of a given age. However, when F(tau, V) is averaged over the age distribution of a population in balanced exponential growth to give the growth rate function f(V), the latter may rise more steeply than V.

Preparation of large quantities of synchronized mammalian cells in late G1 in the pre-DNA replicative phase of the cell cycle.

Abstract Chinese hamster cells, line CHO, can be synchronized in late G1 by growth in isoleucine-deficient medium for 30 h followed by resuspension in fresh, complete medium containing either hydroxyurea (to 10−3 M) or cytosine arabinoside (to 5 μg/ml) for 10 h. Cells which are then washed and resuspended in fresh, complete medium without drugs initiate DNA synthesis shortly thereafter, then commence dividing within 7 h. The technique yields large quantities of cells suitable for biochemical studies of events associated with initiation of genome replication and completion of interphase.

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.

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.