Biomedical subjects

J L Roti Roti

Publications and source records attributed to J L Roti Roti.

At least 19 recordsLinked to original sources

PubMed1996

DNA supercoiling changes and nuclear matrix-associated proteins: possible role in oncogene-mediated radioresistance.

PURPOSE: Transfection with either H-ras or H-ras and c-myc has been shown to confer radioresistance in rat embryonal cells (REC). REC primary, transfected with either c-myc, H-ras or cotransfected with c-myc and H-ras (in ascending order of radioresistance and tumorigenicity), were used as an in vitro model system to determine if nuclear matrix-mediated higher order DNA organization contributes to oncogene-mediated radioresistance. METHODS AND MATERIALS: DNA damage induction and repair were measured by the alkaline and neutral filter elution assays. Analysis of the ability of DNA loop domains to undergo supercoiling changes in the presence of radiation-induced damage was determined by the fluorescent halo assay (FHA). Because DNA loops are organized by the nuclear matrix (NM), a study of NM-associated proteins by high resolution two-dimensional gel electrophoresis was performed. RESULTS: Induction and repair rates of DNA single- and double-strand breaks were similar for the relatively radiosensitive c-myc transfected and the radioresistant c-myc + H-ras transfected cells. However, the degree of inhibition of DNA supercoil rewinding in the presence of radiation-induced damage was less in the radioresistant cells and was inversely correlated with survival. A progressive loss of NM-associated proteins was observed, which correlated with increasing radioresistance and tumorigenicity in these cell lines. In addition, some protein changes were consistent with the possibility that these changes could be involved in DNA anchoring. CONCLUSIONS: Increased radioresistance associated with increasing tumorigencity in these oncogene-transfected cell lines could be due to changes in NM-mediated DNA organization, possibly via differences in NM protein composition that occur following oncogenic transfection.

Animals↗

PubMed1996

Uncoupling of M-phase kinase activation from the completion of S-phase by heat shock.

Chronic exposure of asynchronous HeLa cell cultures to 41.5 degrees C leads to an accumulation of cells in the S-phase, spontaneous premature chromosome condensation, and loss of clonogenicity (M.A. Mackey, S. L. Anolik, and J. L. Roti Roti. Cancer Res., 52: 1101-1106, 1992). In this report, we show that increases in histone H1 kinase activity during 41.5 degrees C exposure occur coincidentally with the appearance of premature chromosome condensation. Furthermore, this kinase activity is shown to be associated with M-phase kinase complexes containing cyclin B1. These increases in the activity of M-phase kinase were found to occur concomitantly with an elevation in cyclin B1 mRNA and an accumulation of cyclin B1 protein. Because cyclin B1 transcription begins in the S-phase, it is probable that the heat-induced delay in the S-phase allows the accumulation of abnormally high cyclin B1 levels. Elevated cyclin B1 levels could then account for the observed abrogation of the cell cycle checkpoint, which usually assures that mitosis does not proceed until DNA replication is complete. This involvement of M-phase kinase in heat-induced cytotoxicity demonstrates the importance of the coordinate regulation of the processes of DNA replication and entry into mitosis.

Cell Cycle↗

PubMed1996

The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2.

Antigens, Neoplasm↗

PubMed1996

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression.

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd) -pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G2, i.e., the fraction of BrdUrd-negative G2 cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G2 and in S are equally delayed. S phase transit of cells irradiated in S or in G1 did not appear to be affected. There were apparent changes in flow of cells out of G1, which could be explained by the delayed entry of G2 cells into the compartment because of G2 arrest. Thus, in asynchronous cells the method was able to detect G2 delay in those cells irradiated in S and G2 phases and demonstrate the absence of cell-cycle delays in other phases.

Animals↗

PubMed1996

DNA supercoiling changes and nucleoid protein composition in a group of L5178Y cells of varying radiosensitivity.

Cell of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-1 and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage.

Animals↗

PubMed1996

Heat-induced modifications in the association of specific proteins with the nuclear matrix.

Nuclei isolated from heat-shocked mammalian cells have an increased protein content which reflects an enhanced protein binding to nuclear structures. These nuclear changes are correlated with cell survival and inhibition of DNA replication, transcription and repair of DNA damage. It appears that most of the altered protein binding occurs in association with the nuclear matrix. The present study was conducted to determine if measurements of specific proteins in isolated nuclei reflect changes that occur at the nuclear matrix. The amounts of various proteins associated with HeLa cell nuclei and nuclear matrices after heat shock were measured by (1) densitometric scans of Coomassie blue-stained gels, (2) immunoblotting with antibodies to nuclear proteins and (3) antisera raised against nuclear matrix proteins from heated cells. These measurements revealed heat-induced increases in the levels of many nuclear matrix proteins. While a number of proteins show similar changes in both nuclei and nuclear matrices, for many the extent of increased association with the nuclear matrix is not reflected in the measured changes in the nuclei. These results are essential for understanding and studying further the relationships between the cellular response to hyperthermia and heat-altered associations of specific proteins with either nuclei or nuclear matrices.

Cell Nucleus↗

PubMed1994

Accelerated G1-transit following transient inhibition of DNA replication is dependent on two processes.

Accelerated G1 transit in HeLa cells develops after a temporary inhibition of DNA synthesis in the previous generation. G1 acceleration has been found to be dependent on (1) the cell-cycle position at the time of the inhibition and (2) the duration of the inhibition. The degree of G1 acceleration correlates with the position in S-phase at which DNA synthesis is blocked. G1 transit time decreases gradually beginning with early S blocks and reaches a minimum when DNA synthesis is inhibited at mid S-phase. A minimum of 4 h continuous inhibition at this position resulted in maximum G1 acceleration. Unbalanced growth, i.e., the nonspecific increase in total protein or RNA levels that results from inhibition of DNA synthesis, did not correlate with the decrease in G1 transit time. On the other hand, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed specific changes in four nuclear proteins of molecular weights 120, 66, 57, and 51 kDa in cells immediately following release from inhibition of DNA synthesis at mid S-phase. A Western blot analysis of cyclins D and E showed an increased accumulation of cyclin E at 0, 1, 5, 7, and 12 h following cell release from the mid S-phase block. Therefore, it is unlikely that changes in the ratio of protein/DNA or RNA/DNA are responsible for the decrease in G1 transit times as suggested by the unbalanced growth model of G1 acceleration. Instead, G1 acceleration may result from the accumulation during S-phase blockage, and subsequent retention through G2 and mitosis, of specific proteins required for cell transit through G1 in the next cell cycle.

Aphidicolin↗

PubMed1994

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

Animals↗

PubMed1994

Heat-induced changes in nuclear-associated proteins in normal and thermotolerant HeLa cells.

In the present study we used one-dimensional 7-10% gradient SDS polyacrylamide gels to resolve the proteins associated with nuclei isolated from normal and heat-shocked cells. By analyzing the relative optical density of 27-30 polypeptide bands as a function of the duration of the heat shock or the time of incubation after heating, we were able to observe the following regarding heat-induced alterations in nuclear protein binding. Various proteins show an increased nuclear association in terms of their nuclear protein content, but the kinetics of this association is not identical for individual proteins. Moreover, the changes in these associations are differentially affected in nuclei from thermotolerant cells. This type of analysis demonstrates the possibility that the cellular effects of hyperthermia could be correlated with the altered binding and/or increased presence of specific proteins associated with the nucleus in heated cells.

Blotting, Western↗

PubMed1994

Heat-shock-induced changes in nuclear protein and cell killing in thermotolerant HeLa cells.

The correlation between the protein content of nuclei and heat-induced cell killing was determined for HeLa cells with various levels of thermotolerance. Thermotolerance was induced by heating cells at 45 degrees C for 15 or 30 min and then incubating them at 37 degrees C for 5 or 24 h. This procedure resulted in four different levels of thermotolerance requiring up to 5 h of heat at 45 degrees C to kill more than 90% of the most resistant cells. Upon exposure to 45 degrees C, the increase in the protein content of isolated nuclei was proportionally less for thermotolerant cells. The difference between the initial increase in nuclear protein content for normal and thermotolerant cells was relatively small for shorter heating times but became clearly evident for longer heating exposures. The correlations between cell killing and nuclear protein content were not statistically different from controls for any of the various levels of thermotolerance. The correlation was measured over survival levels below 0.1 in thermotolerant cells. Because thermotolerant HeLa cells are very resistant to heat, previous studies had not tested the correlation to survival levels below a half-decade of cell killing. These results should resolve some of the conflicting observations reported in the literature and are consistent with the suggestion that heat-induced changes in binding of nuclear protein play a key role in the lethal effects of hyperthermia.

Cell Death↗

PubMed1993

Flow cytometric BrdUrd-pulse-chase study of heat-induced cell-cycle progression delays.

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell-cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43 degrees C for 5, 10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G1, G1S, and G2) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G1 compartment of approximately 16 min per minute of heat at 43 degrees C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43 degrees C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was approximately 25 min per minute of heat at 43 degrees C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G2 + M. The decrease in the rate of transit out of G2 for cells heated in G2 was significantly larger than that for any other compartment, consistent with previous studies, which showed a G2 accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.

Animals↗

PubMed1993

Alterations in nuclear protein mass and macromolecular synthesis following heat shock.

Possible correlations between post-heat alterations in nuclear protein mass and the resumption of macromolecular (DNA, RNA, and protein) synthesis were investigated in CHO cells. Nuclear protein content was measured using flow cytometry. Macromolecular synthesis was measured by incorporation of radioactively labeled precursors into TCA-precipitable material of whole cells and isolated nuclei. Following an initial increase which was dependent on the heat dose, nuclear protein mass decreased following a monotonic function which appeared to be multiphasic. The synthesis of DNA, RNA, and protein was inhibited in a manner dependent on the heat dose and remained suppressed for an interval dependent on the heat dose before recovery. The kinetics of resumption of DNA, RNA, and protein synthesis correlated linearly with the nuclear protein mass measured immediately after heating. Also, the time of onset of recovery of RNA and protein syntheses correlated linearly with the time at which nuclear protein mass returned to 125% of control, a level which has been implicated as a possible threshold in previous studies. More significantly, the onset of the resumption of DNA synthesis showed a one-to-one correlation with the time at which the nuclear protein mass returned to 125% of control. These correlations suggest that there may be causal relationships between the resumption of DNA, RNA, and protein synthesis and the reduction of the amount of nuclear protein binding, particularly in the case of DNA synthesis.

Animals↗

PubMed1992

A model of heat-induced clonogenic cell death.

When cells are exposed to elevated temperatures, clonogenicity decreases in a temperature-dependent manner displaying non-linear kinetics. In this report we show that the survival of synchronized cell populations after heat exposure can be characterized by a parameter epsilon, which is normally-distributed throughout the population. Cells whose corresponding epsilon-value lies below an arbitrary threshold value epsilon min are unable to form a colony. Upon transfer of a cell culture to an elevated temperature, the mean value of the epsilon distribution decreases exponentially over time with a rate dependent upon the difference between the current and final mean value, until reaching a final value epsilon f dependent on the particular temperature used, thus representing a larger proportion of cells with epsilon less than epsilon min which are non-clonogenic. The analyses evaluate a temperature-independent parameter k which is presumed to be dependent upon the growth conditions of the cell population, and a temperature-dependent parameter epsilon f, which is characteristic of the new temperature-perturbed steady state. Comparison of the results of analysis of synchronized G1 cell survival data to that for S phase cells obtained over a range of temperatures shows that it is sufficient to ascribe the increased thermal sensitivity of S phase cells to a different k value, the temperature-dependence of epsilon f being identical in the two cases. Linear regression analysis of the temperature-dependence of epsilon f, when expressed as 1n (-epsilon f) = a0 + a1/T demonstrated strong linearity (r2 = 0.99) for either the individual (G1 or S) or combined data sets. This description of heat-induced cytotoxicity may be of use as the basis for a dose concept for the clinical administration of hyperthermia.

Animals↗

PubMed1992

Cellular mechanisms associated with the lack of chronic thermotolerance expression in HeLa S3 cells.

Chronic thermotolerance is an operational definition for that resistance to cell killing by heat which develops during a protracted exposure at temperatures generally in the range of 41.5-42.5 degrees C which is usually observed as a reduction in the slope of the survival curve. While Chinese hamster ovary (CHO) cells are generally more sensitive to high-temperature heat shock than HeLa cells, studies of cells maintained in suspension culture at 41.5 degrees C demonstrated CHO cells to be more resistant to cell killing at this temperature than HeLa cells, due to the expression of chronic thermotolerance in the hamster cell line and the corresponding lack of chronic thermotolerance expression in the HeLa cell line. Experiments were conducted in the two cell lines while heating under identical conditions, in order to detect any cell line-specific changes in heat-induced perturbation of cell cycle progression and the expression of chronic thermotolerance. Our results showed that CHO cells exhibited a G1 block which lasted throughout the course of the 32-h heating period. HeLa cells, however, failed to accumulate in G1, progressing instead into S phase where spontaneous premature chromosome condensation and nuclear fragmentation were observed. This accumulation of cells with condensed chromatin possessing S-phase DNA content exhibited a linear, one-to-one functional relationship with the fraction of dead cells. Previous studies (M.A. Mackey and W.C. Dewey, Int. J. Hyperthermia, 5:405-415, 1989) demonstrated that synchronized S-phase CHO cells heated at 41.5 degrees C and 42 degrees C were unable to express chronic thermotolerance. Therefore, we hypothesize that progression of cells out of G1 phase into S and G2-M phases leads to lethal processes that prevent the expression of chronic thermotolerance in the HeLa cell line. This hypothesis is strengthened by the observed correlation between the accumulation of "mitotic-like" cells and decreased survival, suggesting that the G1 block observed in CHO cells is causally connected with the expression of chronic thermotolerance.

Animals↗

PubMed1992

Initial characterization of heat-induced excess nuclear proteins in HeLa cells.

Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus.

Cytoskeletal Proteins↗

PubMed1992

Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.

Blotting, Western↗

PubMed1992

Changes in heat and radiation sensitivity during long duration, moderate hyperthermia in HeLa S3 cells.

Step-up heating and thermal radiosensitization were studied at 41.5 degrees C in HeLa S3 cells under conditions where chronic thermotolerance was not expressed. In spite of this lack of thermotolerance expression, it was possible that thermotolerance to higher temperature treatment had developed. Accordingly, cells were incubated for various times at 41.5 degrees C, then immediately shifted up to 45 degrees C, whereupon heating continued for up to 75 min. Thermotolerance to 45 degrees C heating was observed after 8 hr incubation at 41.5 degrees C and decayed by 32 hr of continuous incubation at 41.5 degrees C. When the time of 45 degrees C treatment was extended to 150 min, the biphasic survival response indicated that chronic thermotolerance was expressed at 45 degrees C, even though it was not expressed during the 41.5 degrees C treatment. Thus, chronic thermotolerance can develop under conditions (e.g., at 41.5 degrees C) where it is not expressed, yet be expressed under other conditions (e.g., during 45 degrees C exposure). When cultures were x-irradiated after various periods of 41.5 degrees C treatment, maximum thermal radiosensitization was observed after 4 hr of incubation at 41.5 degrees C, for which no cell killing was observed due to heat alone. The radiosensitization observed decreased the Do and Dq values from about 1.3 Gy to 0.7 Gy and from about 2.0 Gy to 1.0 Gy, respectively. As the duration of the 41.5 degrees C pre-treatment was extended up to 32 hr, no additional thermal-radiosensitization was observed; all killing due to the heat exposure at 41.5 degrees C was additive to the radiation killing after the initial induction of thermal radiosensitization. These results demonstrate differences in the thermal and radiation responses of HeLa cells when compared to earlier studies using CHO cells and may be more relevant to the clinical setting.

Acclimatization↗

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↗