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Biomedical subjects

R Higashikubo

Publications and source records attributed to R Higashikubo.

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↗
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↗
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

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

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↗
PubMed1987

The effect of chromatin decondensation on DNA damage and repair.

The effects of chromatin compaction on X-radiation-induced cell killing and the induction and repair of DNA damage were studied in Chinese hamster ovary cells deprived of isoleucine for 24 h (Ile- cells) and compared to untreated controls. The results show that chromatin is decondensed in Ile- cells; i.e., in Ile- cells the nuclear area occupied by heterochromatin decreased 30-fold over control cells, both the rate and limit of micrococcal nuclease digestion were greater for Ile- cells, and 14.2% more propidium iodide was intercalated into the Ile- cell chromatin. The X-ray-induced cytotoxicity did not change in Ile- cells versus the control cells (D0 = 0.99 Gy) nor did the X-ray-induced DNA damage. However, the repair of DNA damage produced by 10 Gy proceeded with different kinetics in Ile- cells when compared to the controls. The initial rate of DNA damage repair was slower in Ile- cells by a factor of 2 compared to controls (the time required to rejoin 50% of the lesions was 6 versus 3 min, respectively). However, after 2 h of repair no DNA damage was detected in either group. Therefore, we conclude that this decondensation of chromatin, per se, does not directly modify the induction or ultimate repair of DNA damage by X radiation or cell clonogenicity and thus does not appear to be a primary factor in cell survival.

Animals↗
PubMed1986

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein.

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.

Amino Acids↗
PubMed1986

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements.

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.

Cell Cycle↗
PubMed1986

Nuclear protein content and cell progression kinetics following X irradiation.

After irradiation with 4 Gy of X rays the nuclear protein and DNA contents (to determine cell-cycle position) of HeLa cells were determined by isolating nuclei and staining them with the fluorescent dyes fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for DNA. Immediately following irradiation there was no change in the shape of the bivariate (FITC-PI) histogram. At 3 and 4 h after irradiation the region of the histogram which corresponds to mitotic cells had disappeared. At 6 h nuclei reappeared in this region. The maximum rearrangement of the histogram (i.e., maximum accumulation of cells in G2 with minimum cells in G1) occurred at 10.5 h after irradiation, which is later than the time required for mitotic recovery. No change in nuclear protein content of cells in G1 and S was observed. However, beginning at 4 h after irradiation and continuing throughout the period of observation, a small (10-20%) but significant increase in nuclear protein content was observed for nuclei isolated from cells in G2. The increase in nuclear protein content may be part of the mechanism of G2 arrest and/or may reflect unbalanced growth.

Cell Cycle↗
PubMed1985

Quantitation of lymphocyte subsets by immunofluorescence flow cytometry in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDC) is a disease in which immune aberration has been postulated but not confirmed. The frequency of lymphocyte subsets was evaluated in 22 patients with IDC and in 22 blood bank control subjects, using monoclonal antibodies to cell surface markers to allow cell sorting by immunofluorescence flow cytometry. Eighteen patients with heart failure from other causes were also studied. Functional correlations were also made for the natural killer cell subset. Total T-cell frequency, determined with antihuman T (Hybritech), was similar in IDC and control groups: mean 73 +/- 12% in IDC patients and 70 +/- 9% in control subjects. B-cell frequency, determined with antihuman Ia (Hybritech), was also similar: 36 +/- 11% in IDC patients and 31 +/- 10% in control subjects. Helper T-cell frequency, identified by OKT4 (Ortho), averaged 47 +/- 11% in IDC and 44 +/- 8% in control subjects (difference not significant). Suppressor/cytotoxic T-cell frequency, established by OKT8, was the same: 28 +/- 8% in IDC and 30 +/- 7% in control subjects, although relative deficiency in suppressor functional activity has been reported in IDC. Helper to suppressor (OKT4/8) ratios, aberrant in many autoimmune diseases, did not differ significantly (IDC 1.9 +/- 0.8, control 1.5 +/- 0.6). Lymphocyte subsets and OKT4/8 ratio were also similar between IDC and heart failure patients. Natural killer cell frequencies, estimated using 2 antibodies (antihuman Leu-7 and Leu-11, Becton-Dickinson) were the same (9.3 +/- 4.9% in IDC patients, 9.0 +/- 4.5% in control subjects, Leu-7).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

DNase I sensitivity of nuclear DNA measured by flow cytometry.

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Murine mammary tumour cells in vitro. I. The development of a quiescent state.

Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G1 DNA content increasing from 50% in the P state to greater than 97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of greater than or equal to 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since less than 2.5% of the cells incorporated [3H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was greater than 95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents.

Animals↗
PubMed1984

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells.

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.

Animals↗
PubMed1984

Protein cross-migration during isolation of nuclei from mixtures of heated and unheated HeLa cells.

To investigate the cross-migration of proteins during nuclear isolation heated and control cells were mixed prior to nuclear isolation. These nuclei were stained with fluorescein isothiocyanate (FITC, a protein-specific stain) and with propidium iodide (PI, a DNA-specific stain). Flow cytometric (FCM) analysis showed two populations distinguishable on the basis of protein content. The protein content of the nuclei in the upper population was identical to that for nuclei isolated from heated cells while that for the lower population had a protein content identical to the protein content of nuclei from control cells. This result shows that the heat-induced increase in nuclear protein content occurred throughout the entire population of nuclei (i.e., in G1, S, and G2 nuclei) and that the measured protein content of nuclei was not affected by the presence of the other population during isolation. The capability of the FCM to sort subpopulations from different regions of a histogram was used to separate the subpopulations after analysis. When control cells were prelabeled with [3H]leucine and mixed with unlabeled heated cells, 11% of the radioactivity was found to be associated with the nuclei from heated cells. Autoradiographs showed grains over approximately 99% of the nuclei from heated cells. When [3H]TdR was used as a label in a similar experiment, only 0-3% of the label was observed to become associated with the population of nuclei from heated cells and autoradiography showed that 97% of these nuclei were not labeled. Comparable results were obtained when the labeled cells were heated and the control cells were left unlabeled. These results show that a small amount of protein (approximately 10% of the nuclear protein) will cross-migrate during nuclear isolation without affecting the net amount of protein in either population.

Cell Fractionation↗
PubMed1983

Comparison of the cell kill measured by the Hoechst-propidium iodide flow cytometric assay and the colony formation assay.

A flow cytometric live-dead cell assay that uses the dual staining of Hoechst 33342 and propidium iodide (HO-PI) was evaluated for its ability to determine the clonogenicity of treated HeLa cells. The colony-forming assay was used as the reference to determine the capability of the HO-PI assay to measure the proportion of clonogenic cells present in a given population. The viability estimates of both the FCM and trypan blue dye exclusion assays accurately predicted the colony-forming efficiency (CFE) of untreated populations of HeLa cells. However, immediately after treatment with either heat, freeze-thawing, or ionizing radiation the HO-PI assay greatly overestimated the clonogenicity of HeLa cells. This lack of correlation between the FCM determined viability and clonogenic survival was also observed when the cells were heated (45 degrees C, 30 min) and then assayed at 0, 1, 2, 3, 6, 12, 16 or 23 hr after treatment. These data demonstrate that viability as estimated by the HO-PI did not predict survival after acute treatment. In sixty-seven mouse mammary tumour cells, when clonogenicity decreased as a function of time spent in nutrient deprived conditions (chronic treatment), the HO-PI assay again predicted higher CFEs than were measured. Therefore, in these experiments the HO-PI assay could not predict cell death and gave no better measure of cell viability than the trypan blue dye exclusion test.

Benzimidazoles↗