PubMed Health⌕ Search

Biomedical subjects

L J Tolmach

Publications and source records attributed to L J Tolmach.

At least 19 recordsLinked to original sources
PubMed1992

G1 shortening following unbalanced growth: a specific v. nonspecific effect.

The synthesis and abundance of proteins were examined in synchronous populations of HeLa cells under conditions in which the lengthening of S phase, by inhibiting DNA synthesis, resulted in shortening of G1 in the subsequent generation. Mitotically collected cells were resynchronized by incubation with 3 microM aphidicolin from 3 to 12 h after mitotic selection; they were blocked again at various times thereafter to induce unbalanced growth. Cells were labelled with [35S]-methionine before and after release from the block to study the changes in protein synthesis. Triton X-100 soluble and insoluble proteins were analysed by 7-15% gradient SDS-PAGE, and radioactivity incorporation was quantified by liquid-scintillation counting. The degree of G1 shortening correlated with S phase position, increasing gradually from early S and reaching maximum when cells were blocked half-way through S phase. Synthesis of proteins of 120, 66, and 51 kDa was stimulated, and synthesis of a new protein of 57kDa was observed, in cells in which DNA synthesis had been blocked in mid-S. These proteins also showed increased accumulation. These results suggest that the shortening of G1, induced by the prior arrest of cell-cycle progression, is associated with synthesis of specific proteins rather than the non-specific accumulation of cellular proteins through unbalanced growth.

Aphidicolin↗
PubMed1991

Radiation-induced progression delay in HeLa cells blocked in S phase by aphidicolin.

The duration of the cell cycle in synchronous cultures of HeLa S3 cells that were either irradiated with 3.5 Gy of 220-kV X rays in mid-S phase or treated in early G1 or mid-S phase for several hours with 1 or 3 microM aphidicolin, or were subjected to both treatments, was measured by time-lapse cinemicrography. When compared with the generation time of untreated cells, the delay in cell progression with the combined treatment was found to be less than the sum of the delays with the individual treatments, but longer than the imposed delay caused by treatment with aphidicolin alone. Because recovery from potentially lethal radiation damage proceeds in the presence of aphidicolin, this finding suggests that a portion of the radiation-induced delay in cell progression may be associated with processes other than those that directly affect cell viability. It was also observed that the incidence of both spontaneous and radiation-induced sister-cell fusion is decreased in cultures incubated in the presence of aphidicolin.

Aphidicolin↗
PubMed1991

On the identity of the damage expressed by treatment of X-irradiated HeLa cells with different agents.

To determine whether different agents that enhance the expression of potentially lethal X-ray damage (PLD) interact with the same or different lesions (or spectrum of lesions), cell killing was measured in three kinds of experiments: (1) When cells were irradiated in G1 phase and treated with caffeine or hydroxyurea at concentrations that yield maximal response, the same survival plateaus were reached. (2) Treatment of cells irradiated in G1 phase either with caffeine or with hydroxyurea so as to yield survival levels that differed twofold after 4 h incubation, followed by treatment with caffeine to allow expression of PLD in G2 phase, resulted eventually in the same level of survival. (3) When cells were irradiated and treated with caffeine, hydroxyurea, or 9-beta-D-arabinofuranosyladenine (araA) after progressively longer delays, to trace the time course of recovery from the PLD, the responses obtained with caffeine and araA were similar; sensitivity to hydroxyurea was lost more rapidly. The results are consistent with the possibility that these three agents interact with the same lesions, but that different steps in the repair process are inhibited by caffeine or araA than by hydroxyurea.

Caffeine↗
PubMed1990

Kinetics of the development of accelerated cell-cycle transit resulting from inhibition of DNA replication in the previous cycle.

Shortening of the generation cycle in cells in which DNA synthesis had been temporarily inhibited in the previous generation, which has been reported several times in recent years, has been confirmed in HeLa cells. As in the previous studies, the shortening is attributable to accelerated transit of G1 resulting from the accumulation, during the inhibition, of a factor needed for initiation of DNA replication. It is shown that partial (85-96%) inhibition with any one of three inhibitors is effective when the inhibitor is added in G1 or in S, but more complete (99%) inhibition is effective only if the inhibitor is added after cells have entered S. In addition, cells begin to respond to the inhibition after a lag that increases as DNA synthesis in the early part of S is progressively inhibited with aphidicolin, indicating that competence to respond is achieved only after cells have reached a particular point in the replication of their genome.

Antineoplastic Agents↗
PubMed1990

The action of caffeine on X-irradiated HeLa cells. IX. Hypothermic effects.

Hypothermic enhancement of the lethal effect of 3.5 Gy of 220-kV X rays in the absence of caffeine as well as in its presence (4 mM) was examined at temperatures between 10 and 34 degrees C in monolayer cultures in the G1 phase of the cell cycle. Correction has been made for the toxicity of low temperatures, and of caffeine at low temperatures, by concomitantly measuring cell killing in unirradiated cells. In the absence of caffeine, incubation of irradiated cells for up to 34 h at temperatures in the range 15 to 30 degrees C (or possibly 34 degrees C) enhances killing compared to that observed at 38 degrees C; the amount of enhancement is about the same throughout this range, but is nil at 10 degrees C. The enhanced killing induced by caffeine at 38 degrees C decreases as the temperature is lowered to 15 degrees C; there is no enhancement at 10 degrees C. Less killing is manifested in the range 15 to 25 degrees C in the presence of caffeine than in its absence. Recovery (loss of sensitivity to caffeine) and fixation of potentially lethal damage were studied in late-S/G2-phase cells at reduced temperatures by delaying treatment with caffeine for increasing times after irradiation. As the temperature is progressively lowered to 20 degrees C, less recovery is manifested after 5 h of incubation; no recovery is detected in the range 10 to 20 degrees C. Despite extensive recovery at 34 degrees C, no fixation is observed at that (or any lower) temperature in G2-phase cells: the cells are able to recover essentially fully when returned to 38 degrees C. In addition, responses of unirradiated control series to incubation at low temperatures appear to differ from those reported by others for longer treatment times of different cell systems.

Caffeine↗
PubMed1990

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine.

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.

Bromodeoxyuridine↗
PubMed1988

Caffeine-induced modulation of the lethal action of X rays on Chinese hamster V79 cells.

Caffeine-mediated enhancement of the killing of V79 cells by 220-kV X rays at various times in the cell cycle was compared with that of HeLa cells by measuring (i) the dependence of cell survival on the duration of treatment with 5-10 mM caffeine, (ii) the effect of caffeine treatment on the X-ray dose-survival curve, and (iii) the loss of sensitivity to caffeine as a function of time after irradiation. The behavior of V79, while similar in many respects to that of HeLa (reported previously), differs in several ways. Caffeine treatment causes rapid killing immediately after irradiation irrespective of cell age, while HeLa is refractory in S phase and highly sensitive in G2. As with HeLa, the (multitarget) dose-survival curve parameters are reduced by caffeine treatment, but the age-dependent fluctuations in D0 are not eliminated as completely as with HeLa and the extrapolation number assumes values less than unity in the latter part of the cycle rather than in the early part. Loss of sensitivity to caffeine after irradiation early in the cycle appears to undergo a transient reversal in the middle of the cycle, a phenomenon not observed in HeLa.

Animals↗
PubMed1986

The action of caffeine on X-irradiated HeLa cells. VIII. Recovery from potentially lethal damage.

Recovery from potentially lethal radiation damage in HeLa S3 cells has been studied by irradiating synchronous cultures with 4 Gy at selected ages in the cell cycle, initiating treatment with 4 mM caffeine, which prevents recovery, at progressively later times up to 24-30 h after irradiation, and determining the plateau level of survival after incubation with the caffeine until 36-40 h after mitotic collection. Cell recovery appears to begin immediately after irradiation at any time during interphase: an accelerating increase in survival gives way after several hours to a linear increase which lasts for an additional several hours. The median recovery time is approximately 13 h after irradiation at any time during G1, but is markedly shorter (5-7 h) after irradiation in S or G2. The rate of recovery is slightly depressed if DNA replication is inhibited with aphidicolin after irradiation and slightly enhanced if protein synthesis is inhibited with cycloheximide. Both the rate and the extent of recovery are dependent on the location of the cells in the cycle at the time of irradiation--both functions increasing with cell age from the beginning of S, but having different age dependencies in G1. Blocking cell progression with a DNA-synthesis inhibitor before irradiation halts the age-dependent changes.

Caffeine↗
PubMed1984

The action of caffeine on X-irradiated HeLa cells. VI. Damping of the structured age-survival function.

Postirradiation treatment of synchronous HeLa S3 cultures with 4 mM caffeine until greater than or equal to 32 hr after mitotic collection, following exposure to 220-kV X rays at various times during interphase, severely damps the fluctuations in the age-survival curve. Not only does the dose-survival curve essentially lose its shoulder, as reported previously, but it becomes steeper and displays a virtually age-independent terminal slope (D0 congruent to 0.5 Gy). It becomes multicomponent, at least early in the cycle. The residual structure in the interphase age-survival curve, if any, appears to reflect mainly an age-dependent fluctuation in the size of a subpopulation of cells having marked sensitivity to X rays (D0 congruent to 0.25 Gy), though there might be small residual fluctuations in the size of the shoulder and the slope. Mitotic cells also respond to postirradiation treatment with caffeine; they yield a dose-survival curve whose slope is similar to that of the sensitive subpopulation seen in interphase. These findings indicate that most of the structure in the unperturbed age-survival function derives from repair of potentially lethal radiation damage.

Caffeine↗
PubMed1984

The action of caffeine on X-irradiated HeLa cells. VII. Evidence that caffeine enhances expression of potentially lethal radiation damage.

HeLa cells irradiated with 2 Gy of 220-kV X rays suffer a 60-70% loss of colony-forming ability which is increased to 90% by postirradiation treatment with 10 mM caffeine for 6 hr. The detailed postirradiation patterns of cell death and sister-cell fusion in such cultures and in cultures in which the colony-forming ability was brought to about the same level by treatment with a larger (4 Gy) X-ray dose alone or by longer (48 hr) treatment with 10 mM caffeine alone were recorded by time-lapse cinemicrography. Because the patterns of cell death and fusion differ radically in irradiated and in caffeine-treated cultures, the response of the additional cells killed by the combined treatment can be identified as X-ray induced rather than caffeine induced. The appearance of cultures after several days of incubation confirms the similarity of the post-treatment patterns of proliferation in cultures suffering enhanced killing to those occurring in cultures treated with larger doses of X rays alone. It is concluded that X rays do not sensitize cells to caffeine, but rather that caffeine enhances the expression of potentially lethal radiation-induced damage.

Caffeine↗
PubMed1983

Synergistic killing of HeLa cells by hydroxyurea and caffeine.

Synchronous populations of HeLa S3 cells suffer synergistic killing during S phase in the presence of 0.5-5 mM hydroxyurea together with 5-10 mM caffeine. Both the rate and the extent of killing are greater than expected for independent action of the two drugs. Only simultaneous treatment is effective. The dependence of the synergistic killing on cell age resembles the age dependence for killing by hydroxyurea alone (greater than 3 mM), but not that by high concentrations of caffeine. In addition, rapid killing occurs if caffeine is added to cultures that have been incubated in the presence of hydroxyurea from early G1 and are blocked at the beginning of S, although such cells are killed only slowly on continued incubation in greater than or equal to 10 mM hydroxyurea alone. Furthermore, cells that are incubated with the two drugs from early G1 begin to undergo synergistic killing at about 12 h after mitotic collection, but they do not commence DNA replication for another 2-3 h if the drugs are removed. It is concluded that cells that have reached a point in the cycle identical with or close to the end of G1 are sensitive to the combination whether or not they are able to synthesize DNA, and whether or not they are sensitive to hydroxyurea alone. A tentative model is proposed: hydroxyurea is postulated to kill cells by interacting with sites of replication in DNA, and the synergism is attributed to the extra replication points that caffeine is known to induce.

Caffeine↗
PubMed1982

Growth and death of HeLa cells in the presence of caffeine.

Proliferation and death were measured in synchronously growing cultures of HeLa S3 cells during treatment with up to 30 mM caffeine. Changes in the number of colony-forming cells were determined by single-cell plating, while changes in the total number of cells were measured both by electronic counting and by monitoring cell division and physiological death cinemicrographically. At concentrations between 2 and 5 mM, cell killing occurs over several days during which the cells traverse the generation cycle once or a few times before losing colony-forming ability, with consequent proliferation of non-colony-forming cells. This indicates that lethal damage is accumulated with time. Death occurs more rapidly at higher concentrations, without proliferation, the kinetics of inactivation being strongly dependent on the phase of the cycle (cell age) at which treatment is initiated. G1 cells are killed more slowly in 10 mM caffeine than are S cells, but G1 cells respond rapidly to 20 mM caffeine, suggesting the inception of an additional mode of killing. The incidence of sister-cell fusion increases with increasing caffeine concentration above 1 mM. On addition of 10 mM caffeine to a culture prepared from collected mitotic cells, the cells undergo a transient rounding and then respread after several hours; with 20 mM, they never respread. The generation cycle is prolonged in a concentration-dependent fashion, as is the duration of G1; the generation time is doubled in 5-6 mM caffeine. G2 and M are also prolonged at concentrations above 3 mM, but S is not prolonged even by 10 mM caffeine.

Caffeine↗
PubMed1982

Multiplexed time-lapse photomicrography of cultured cells.

A system of cinemicrography has been developed in which a single microscope and 16 mm camera are multiplexed to produce a time-lapse photographic record of many fields simultaneously. The field coordinates and focus are selected via a control console and entered into the memory of a dedicated microcomputer; they are then automatically recalled in sequence, thus permitting the photographing of additional fields in the interval between exposures of any given field. Sequential exposures of each field are isolated in separate sections of the film by means of a specially designed random-access camera that is also controlled by the microcomputer. The need to unscramble frames is thereby avoided, and the developed film can be directly analysed.

Animals↗