Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Proliferative quiescence of normal mast cells resembles that of cold-sensitive mutant mastocytoma cells. Dominant expression of the quiescent state in heterokaryons

Normal murine peritoneal mast cells were fused to serum-deprived, non-proliferating cells of a cultured subline (41-SB-4) of the P-815 murine mastocytoma. Upon reincubation in medium containing 10% horse serum for 48 h, mono- and binuclear 41-SB-4 cells reentered S phase of the cell cycle, while mast cell X 41-SB-4 heterokaryons as well as mono- and binuclear mast cells remained in proliferative quiescence, indicating dominant expression of the quiescent state of mast cells. The quiescent state of normal mast cells thus resembles that of cold-sensitive (cs) mutant cells (21-F) of the undifferentiated P-815 mastocytoma: at the non-permissive temperature of 33 degrees C, the 21-F cells were found to enter a state of quiescence which is characterized by its dominant expression in heterokaryons and by morphological differentiation with the formation of metachromatically staining granules similar to those of mast cells. This suggests that the cellular control mechanisms involved in entry into proliferative quiescence and in morphological differentiation of cs 21-F cells may be analogous to those of normal mast cells and/or their precursors.     

Control of DNA polymerase alpha, beta and gamma activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 degrees C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 degrees C, a transient increase in both cytochrome c oxidase and DNA polymerase gamma was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular cross-section as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 micrograms/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 degrees C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 degrees C or during treatment with 1 mM butyrate, suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in 'gently lysed cells' (obtained by treatment with 0.01% Brij-58) or 'highly lysed cells' (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3H]dTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extract reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants.

Herpes simplex virus type 1 DNA synthesis and infections progeny production were studied in five different conditional hamster (BHK-21) cell cycle mutants. At the nonpermissive temperature (39.5 degrees C), both events were strongly inhibited in four of these cell lines. The degree of inhibition was a reproducible characteristic of each cell mutant and in two cases was dependent upon the multiplicity of infection. Experiments involving shifts to the nonpermissive temperature at least 3 h postinfection at 33.5 degrees C suggested that the defects in viral replication were not due to faulty adsorption, penetration, or uncoating, whereas experiments involving shifts of infected cells from the nonpermissive temperature to 33.5 degrees C revealed the reversible nature of the inhibition.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Control of DNA polymerase α, β and γ activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G₁ at 39.5°C) and two cold-sensitive (arrested in G₁ at 33°C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase α, β and γ activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase α activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase α activities in arrested cells were as high as 15–40% of control values. After return of arrested cells to the permissive temperature, polymerase α activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77–85), the decrease of polymerase α during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase α is tightly coupled with reentry of cells into S phase. For DNA polymerase β and γ activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [³H]dTTP efficiently, while highly lysed cells exhibited a low level of [³H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Isolation of ts mutant cells which arrest in G1/G0 phase at the non-permissive temperature in the presence of appropriate growth factors from a Fischer rat cell line, 3Y1

Two types of cell-cycle-ts mutants were isolated from Fischer rat cell line, 3Y1, and characterized. Clones in one complementation group, tsJT51 and tsJT341, grew at 34 degrees C in the presence of 10% fetal bovine serum (FBS). When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the presence of both FBS and epidermal growth factor (EGF), but died in the presence of one of these growth factors. The cells in the other complementation group, tsJT59, tsJT308, tsJT314 and tsJT349, grew at 34 degrees C in the presence of 10% FBS. When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the simultaneous presence of FBS, EGF and insulin, but died quickly if one of these growth factors was lacking. Growth-arrested cells at 39.5 degrees C were viable at least one or two weeks and had a potency to resume growth following the shift-down of temperature. Those are assumed to be ts mutant cells which enter and stay in G1/G0 phase from the cell cycle at the non-permissive temperature only in the presence of appropriate growth factors.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.     

Defect in prereplicative phase of G0-specific ts mutant, tsJT60

A temperature-sensitive mutant, tsJT60, grew exponentially at both 34 degrees and 39.5 degrees C, but when stimulated from the resting state it entered S phase at 34 degrees but not at 39.5 degrees C. The mutated function appeared to be a prerequisite throughout from 0 to 9 h following the stimulation, in order that G0-arrested cells would enter S phase. When the arrested cells were stimulated with serum, the amount of and synthesis of protein increased at 34 degrees but not at 39.5 degrees C. The amount of polysome fraction was much smaller in stimulated and unstimulated cells at 39.5 degrees C than in those stimulated at 34 degrees C. Of the events reported to increase shortly after the stimulation, uridine transport increased at both temperatures. Mutation in tsJT60 cells may be concerned with the function prerequisite to induce protein synthesis following serum stimulation, resulting in the blocking of cell cycle progression toward S phase at 39.5 degrees C.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.