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J L Roti Roti

Publications and source records attributed to J L Roti Roti.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The suppression of the synthesis of a nuclear protein in cells blocked in G2 phase: identification of NP-170 as topoisomerase II.

Previous studies of a nuclear protein of molecular weight 170 kDa (NP-170) have shown it to have two interesting properties. First, NP-170 synthesis began in mid- to late S phase and became maximal in G2 phase. Second, the synthesis of NP-170 was suppressed in cells blocked in G2 phase following irradiation with 6.8 Gy (J. M. Holland et al., Radiat. Res. 122, 197-208, 1990). The molecular weight of NP-170 is the same as that of Topoisomerase II (Topo II), an enzyme involved in the alteration of DNA supercoiling status with a double-strand passing function. This study was undertaken to determine whether NP-170 could be Topo II. The results from the present study show that both the proteins have identical cell cycle synthesis patterns. The synthesis of both these proteins is suppressed following irradiation. NP-170 was found to be recognized by a Topo II antibody in both Western blots and immunoprecipitation. This study characterizes NP-170 as Topo II.

Autoradiography↗
PubMed1991

Reproductive integrity of mammalian cells exposed to power frequency electromagnetic fields.

Human lymphocytes and Chinese hamster ovary (CHO) fibroblasts were analyzed for cytogenetic and cytotoxic endpoints to determine whether exposure to power frequency (60 Hz) electromagnetic fields (EMF) interferes with normal cell growth and reproduction. An exposure chamber was built to apply variable electric current densities of 3, 30, 300, and 3,000 microA/cm2, simultaneously with a fixed magnetic field of 2.2 G to proliferating cells. The current densities were chosen to bracket those that may be induced in the human body by fields measured beneath high voltage (765 kV) power transmission lines. The electric current was applied through the media of a cell culture chamber positioned between two stainless steel electrodes but separated from direct contact with the culture media by a salt bridge composed of a 1% agarose gel. The magnetic field was generated using two pairs of Helmholtz coils driven 73 degrees out of phase producing an elliptically polarized magnetic field 36 degrees out of phase with the electric field. The EMFs were measured and mapped inside the cell culture chamber to insure their uniformity. CHO cells were exposed continuously for 24-96 hr (depending on experiment) and human lymphocytes were exposed continuously for 72 hr. The EMFs were monitored throughout the entire treatment period using a multichannel chart recorder to verify continuous application of the desired fields. Sister-chromatid exchange and micronuclei were monitored to evaluate the potential for genotoxicity. In addition, standard growth curves, clonogenicity, and cell cycle kinetics were analyzed to evaluate possible cytotoxic effects. The experimental data consistently showed that the growth rate and reproductive integrity of both cell types was unaffected by exposure to the electromagnetic fields.

Animals↗
PubMed1990

Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content.

Two FCM methods utilizing isolated nuclei were described. A DNase I sensitivity assay, employing changes in binding and digestion kinetics of the enzyme as well as the binding of intercalating fluorochrome was used to observe structural changes of chromatin rendered by physical and chemical agents. A nuclear DNA-protein staining method was used to study changes in nuclear protein content, redistribution of populations in the cell cycle, and unbalanced growth, manifested as an extraordinary accumulation of nuclear protein brought about by physical and chemical perturbation.

Animals↗
PubMed1990

Fluorescent methods for studying subnuclear particles.

Fluorescence assays can be used to reveal molecular interactions through rapidly demonstrable particle-associated events. The additional fact that in many cases fluorescent particles may be analyzed on a per-event basis lends credence to such techniques as probes for biologically significant perturbations and their resolution. Perhaps more importantly, the sorting capability of the flow cytometer enables detailed study of these events in cells in relation to their positions in the cell cycle. Further studies on the effect of drugs and other modalities on the organization of the genome and the nuclear matrix should prove of interest because the interactions of chromatin and this subnuclear particle could be predictive of the state of DNA metabolism under such conditions. With the additional ability of following such organizational changes through the cell cycle, the mechanisms of reversal of perturbing events might be elucidated.

Cell Cycle↗
PubMed1990

Repair of radiation-induced DNA damage in thermotolerant and nonthermotolerant HeLa cells.

The effect of heat exposure on the repair of radiation-induced DNA damage which inhibits the ability of nuclear DNA to undergo supercoiling changes was studied using the fluorescent halo assay in thermotolerant and nonthermotolerant (normal) cells. The assay utilizes an intercalating, fluorescent dye to unwind and rewind endogenous DNA supercoils. When HeLa cells are exposed to 17.3 Gy radiation the ability of DNA to be rewound into supercoils is completely inhibited. However, the ability of DNA to rewind is 70% restored by 30 min after irradiation. Both thermotolerant and normal cells exposed to 45 degrees C for 30 min prior to irradiation had a rewinding ability intermediate between control and unheated cells, but there was no restoration of rewinding ability up to 3 h postirradiation. Thus, when irradiation immediately followed heating, there was no difference between thermotolerant and normal cells. However, when various time intervals were imposed between heating and irradiation, a difference in the ability of the cells to recover from heat-induced alterations became apparent. In normal cells after 6 h of postheat incubation the cells' ability to restore DNA supercoiling was approximately the same as that of control cells, while in thermotolerant cells only 2 h was required to repair the ability to restore supercoiling at the same rate. The rate of repair of DNA remained correlated with relative nuclear protein content as measured by fluorescein isothiocyanate staining in both thermotolerant and normal cells, indicating a possible relationship between the two.

Acclimatization↗
PubMed1990

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle.

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.

Caffeine↗
PubMed1989

Flow cytometric studies of the nuclear matrix.

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.

Cell Nucleus↗
PubMed1989

Changes in the structure of nucleoids isolated from heat-shocked HeLa cells.

Using a technique to detect changes in DNA supercoiling which allows one to visualize both DNA unwinding and rewinding in presence of the intercalating dye, propidium iodide (PI), we showed that hyperthermic treatment (30 min at 45 degrees C) of HeLa S3 cells alters the response to the intercalating dye. Depending on the treatment conditions, we observed a reduction in the maximum size of the DNA loop that can be measured at the relaxation point (PI concentration 5-7.5 micrograms/ml). Cellular heating also affected all degrees of DNA rewinding (measured as a function of PI concentrations between 10 and 50 micrograms/ml). By 6 h after cellular heating these heat effects had disappeared. This time interval correlated with the time necessary for recovery from a heat-induced increase to normal nuclear and nucleoid protein content. Using gel electrophoresis we showed that the nucleoids (DNA plus nuclear matrix proteins) after heat exposure are enriched in several polypeptides and that there is a specific increase in HSP 72/73. We hypothesize that the altered response to the intercalating dye after cellular heat shock is due to an increase in polypeptides associated with the nuclear matrix thereby altering the DNA-nuclear protein matrix anchor points.

DNA, Superhelical↗
PubMed1989

Comparative effects of caffeine on radiation- and heat-induced alterations in cell cycle progression.

The effects of 3 mM caffeine on cell cycle progression of HeLa S3 cells exponentially and asynchronously growing in suspension culture were studied following exposure to 6.8 Gy gamma irradiation or 30 min at 45 degrees C hyperthermia. The stathmokinetic method, in which cells are grown in the presence of colcemid for the duration of experiment, in combination with two flow cytometric techniques, propidium iodide staining of DNA and acridine orange staining following acid denaturation of chromatin, were used to determine the fraction of cells in four cell cycle compartments, G1, S, G2, and M. Radiation and caffeine acted in a complementary manner, in which radiation reduced the caffeine-induced delays in cell cycle progression and caffeine prevented completely the radiation-induced accumulation of cells in G2 and mitotic delay. Heat and caffeine had additive effects on alterations in cell cycle progression. Cells containing spontaneous prematurely condensed chromatin were observed transiently immediately following heat exposure. These cells appeared to be in G2 and late S phase.

Caffeine↗
PubMed1989

DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells.

DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.

Animals↗
PubMed1989

The relationship of increased nuclear protein content induced by hyperthermia to killing of HeLa S3 cells.

The role of a heat-induced increase in nuclear protein mass in killing of cells by hyperthermia was investigated jointly by two groups that had previously reported apparently conflicting results. A correlation between the fraction of HeLa S3 cells killed and the protein content of nuclei isolated immediately after heat exposure was found. This correlation held when thermal sensitivity was modified by the sensitizers 0.41 M ethanol and 5 mM procaine or by the protector 0.6 M glycerol. However, when the HeLa cells were made thermotolerant by a priming heat exposure of 15 min at 45 degrees C followed by 5 h at 37 degrees C, the correlation no longer held. At the 10% survival level a 1.27-fold greater nuclear protein content was observed in tolerant cells relative to nontolerant cells. Thus no general correlation between initial heat-induced nuclear protein mass changes and hyperthermic cell killing exists. When heated cells were returned to 37 degrees C, a time-dependent reduction in the protein content was observed in nuclei isolated after incubation for various times at 37 degrees C. This rate of reduction in nuclear protein content was found to be accelerated in the tolerant cells. Heat-induced changes in cell-cycle progression had no significant effects on the data obtained. It is concluded from the total data base that not only the absolute increment in nuclear protein mass must be taken into account but also the duration of the binding expressed in the rate of recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗