Biomedical subjects

M Dubsky

Publications and source records attributed to M Dubsky.

8 recordsLinked to original sources

PubMed1986

DNA synthesis in BalB/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase.

ts 2 BalB/C-3T3 mouse fibroblasts are cdc mutants, which arrest late in G1, at or near the G1/S traverse, upon full expression of the heat-sensitive lesion. The kinetics of temperature inhibition of DNA synthesis in logarithmically growing cultures reveal three stages of heat inactivation. During the first generation time equivalent, normal semiconservative, semidiscontinuous replication proceeds but is reduced as cells exit and do not reenter S phase. During a second such period, a minimal rate of normal DNA synthesis is maintained. Thereafter, as the cells move into a third aborted cell division cycle, the rate of DNA synthesis increases. However, all semiconservative synthesis is then replaced by DNA repair replication. Temperature inactivation of the ts 2 protein results in shutdown of nuclear DNA synthesis. In contrast, normal replication of mitochondrial DNA proceeds at control rate throughout the first stage of temperature inactivation. Synthesis of this organellar genome is quantitatively reduced as the cells move into the second phase of heat inhibition. Titration of chromatin-bound DNA with ethidium bromide revealed that wild-type cells exhibit a changing DNA topology as the temperature is raised. Temperature-inactivated ts 2 cells behave as though their DNA has been topologically frozen in the configuration of control cells at or near entry into S phase.

Animals↗

PubMed1985

Cell cycle arrest of heat-inactivated ts 2 BALB/c-3T3 mouse fibroblasts, temperature-sensitive for DNA synthesis.

The ts 2 derivative of BALB/c-3T3 mouse fibroblasts is a cell division cycle (cdc) mutant. Upon expression of the heat-sensitive defect, ts 2 cells arrest late in G1 at, or very near the G1/S traverse. This conclusion derives from three kinds of experiment. In the first the cells were brought to different stages of the cell cycle by physiological manipulation, or with specific anti-metabolites. They were then released from the resulting blocks, and their subsequent cell-cycle progression, at the permissive- and non-permissive temperature (npt), was followed. The second experiment was an execution point analysis. In the third, premature chromosome condensation was performed between metaphase HeLa cells and temperature-blocked ts 2 cells. The resulting prematurely-condensed chromosomes were largely of the morphotype of very late G1 cells. The ts 2 cells are prevented from expressing their defect by temporary incubation at 38.5 degrees C in the G0, non-cycling state and by prior arrest in early S phase, imposed by hydroxyurea treatment. Such prevention is not allowed ts 2 cells incubated at the npt in the absence of isoleucine, a procedure which brings cells to mid-G1 arrest.

Animals↗

PubMed1985

Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

Adenoviridae↗

PubMed1980

Relationship between chromatin structure and replication in mouse L-cells.

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.

Animals↗

PubMed1978

Some properties of chromatin synthesized by mouse-L-cells temperature-sensitive in DNA replication.

When ts A1S9 mouse L-cells are incubated at the nonpermissive temperature (38.5 degrees) DNA synthesis proceeds at the normal rate for 6 to 8 h; it then declines to attain 1 to 5% of this rate after 24 h. General protein synthesis from precursor leucine is relatively unaffected by the high temperature. In contrast, protein formation from lysine (and arginine) remains unchanged for 12 to 15 h after temperature upshift. It then drops and plateaus at about 25% of the initial rate after 32 h. The chromatin protein and DNA are fully conserved in ts A1S9 cells incubated at 38.5 degrees for at least 24 h after full expression of the ts defect. Temperature inactivation of the ts A1S9 gene product results in inhibition of de novo formation of chromatin. This is evidenced by coordinate suppression of incorporation of dThd and of lysine and arginine into chromatin-bound DNA and histone, respectively.

Chromatin↗

PubMed1978

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication.

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.

Cell Count↗

PubMed1977

Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication.

Temperature-sensitive (ts) A 1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 degrees C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6-8 h after the cells are shifted to 38.5 degrees C, and then declines to reach a minimum at 20-24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.

Animals↗

PubMedDate not supplied

Toremifene resistance in a rat mammary tumour model.

The effect of Toremifene on cell growth in vitro was tested on the R3230AC rat mammary adenocarcinoma as model. Toremifene differed from Tamoxifen in that it did not induce resistance; some cross-resistance was observed in Tamoxifen tolerant tumour cell lines. Toremifene does not influence P-glycoprotein expression in this model and at the levels studied.

ATP Binding Cassette Transporter, Subfamily B, Mem↗