Epidermal growth factor has a unique effect in combination with fetal bovine serum to bypass the ts-block of G0-specific ts mutant tsJT60

tsJT60 cells are G0-specific temperature-sensitive mutants of the cell cycle from Fischer rats i.e., they grow exponentially at both 34 degrees and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS) from the resting state (G0) they enter S phase at 34 degrees C but not at 39.5 degrees C. Epidermal growth factor (EGF) also induced DNA synthesis, although weakly, in G0-arrested tsJT60 cells at 34 degrees C but failed at 39.5 degrees C. When G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus EGF, they entered S phase and divided. Somatomedin C, insulin, or transferrin had a weak effect in inducing DNA synthesis in G0-arrested cells when applied at 34 degrees C or with FBS at 39.5 degrees C. Fibroblast growth factor, platelet-derived growth factor, or 12-O-tetradecanoylphorbol 13-acetate had no such stimulatory effect at 39.5 degrees C. Binding of 125I-somatomedin C was not temperature-sensitive. Several other ts mutant cells that were blocked at 39.5 degrees C from entering S phase from the resting state following FBS addition were stimulated by FBS plus EGF at 34 degrees C but not at 39.5 degrees C.     

Bypass of the ts block of tsJT60, a G0-specific ts mutant from rat fibroblasts, by fetal bovine serum and epidermal growth factor

tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.     

tsJT60, a cell cycle G0-ts mutant, becomes lethal at non-permissive temperature by transformation with adenovirus 5 when the expression of E1B gene is lacking

tsJT60, a temperature-sensitive (ts) mutant cell line of Fischer rat, is viable at both permissive (34 degrees C) and non-permissive (39.5 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with fetal bovine serum (FBS) from G0 phase they re-enter S phase at 34 degrees C but not at 39.5 degrees. When tsJT60 cells were transformed with adenovirus (Ad) 5 wild type, they grew well at both temperatures, expressed E1A and E1B genes, and formed colonies in soft agar. When tsJT60 cells were transformed with Ad5 dl313, that lacks E1B gene, the transformed cells grew well at 34 degrees C but failed to form colony in soft agar. They died very soon at 39.5 degrees C. 3Y1 cells (a parental line of tsJT60) transformed with dl313 grew well at both temperatures, although neither expressed E1B gene nor formed colonies in soft agar. The phenotype of being lethal at 39.5 degrees C of dl313-transformed tsJT60 cells was complemented by cell fusion with 3Y1BUr cells (5-BrdU-resistant 3Y1), but not with tsJT60TGr cells (6-thioguanine resistant tsJT60). These results indicate that the lethal phenotype is related to the ts mutation of tsJT60 cells and also to the deletion of E1B gene of Ad5.     

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.     

Induction of cellular DNA synthesis in G0-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses

tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.     

Isolation of a G0-specific ts mutant from a Fischer rat cell line, 3Y1

A ts mutant clone, tsJT60, was isolated from Fisher rat cell line, 3Y1. During the exponential growth at both 34 and 39.5 degrees C, tsJT60 did not appear as ts mutant cells. However, once entered resting state (G0) under serum deprivation at the confluent state, they could re-enter S phase at 34 degrees C but could not at 39.5 degrees C following the stimulation of cells either by the addition of fetal bovine serum or by trypsinization and replating. These and other results suggested that tsJT60 is a G0-specific ts mutant, i.e., the cells have ts defect(s) in the function which is required for the stimulation from the resting state to S phase but not for the progression of the cell cycle in an exponential growth phase.     

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.     

A cell cycle ts mutant, tsJT16, is defective in p70 synthesis through protein kinase C-dependent and -independent pathways.

tsJT16 is a G0/G1 ts mutant from the Fischer rat fibroblast line. It has a ts defect in a function operating early after growth stimulation with fetal bovine serum (FBS). A primarily induced gene product, p70, was not synthesized at 40 degrees C after stimulation with serum, while c-fos and c-myc mRNAs accumulated under the same condition. This paper reports that p70 was synthesized following stimulation of G0-arrested cells with platelet-derived growth factor, epidermal growth factor (EGF), and 12-0-tetradecanoylphorbol-13-acetate (TPA) at 34 degrees C, but not at 40 degrees C. However, it was synthesized at both temperatures after addition of A23187. In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34 degrees C, whereas TPA did not. Fibroblast growth factor and insulin did not induce p70. Induction of c-fos and c-myc occurred at both temperatures after the stimulation with FBS, TPA or A23187. These results indicated that the defect in tsJT16 to induce p70 is likely to be located at the common downstream of protein kinase C-dependent and -independent pathways, but is independent from the pathway of calcium mobilization.     

A cell cycle G0-ts mutant, tsJT60, becomes lethal at the nonpermissive temperature after transformation with adenovirus 12 E1B 19K mutant

tsJT60, a temperature-sensitive (ts) cell-cycle mutant of Fischer rats, is viable at both the permissive (34 degrees C) and nonpermissive (40 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with serum from G0 phase they enter S phase at 34 degrees C but not at 40 degrees C. tsJT60 cells transformed with human adenovirus (Ad) 12 dl205, which lacks the E1B 19-kDa polypeptide gene, were lethal at 40 degrees C, whereas tsJT60 cells transformed with Ad12 wt, dl207, which lacks E1B 58-kDa protein gene, or in206B, which produces 19- to 58- kDa fused protein, were viable. Degradation of cell DNA occurred in dl205-transformed tsJT60 cultured at both 34 degrees C and 40 degrees C. Neither cytocidal phenotype nor degradation of DNA occurred in 3Y1 cells (a parental line of tsJT60) transformed with dl205. These results suggest that the lethal phenotype and degradation of DNA are related to the ts mutation in tsJT60 and also to the lack of Ad12 E1B 19kDa polypeptide.     

Expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive mutant of the cell cycle

We have investigated the expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive (ts) mutant of Fischer rat cells, which, on the basis of its kinetic behavior, can be classified as a G0 mutant. It grows normally at 34 degrees C and also at 39.5 degrees C if shifted to the higher temperature during exponential growth. However, if the cell population is first made quiescent by serum deprivation, subsequent stimulation by serum induces the cells to enter S phase at 34 degrees C but not at 39.5 degrees C. A panel of growth-regulated genes was used that included three protooncogenes (c-fos, c-myc, and p53), several genes that are induced in G0 cells stimulated by growth factors (beta-actin, 2A9, 2F1, vimentin, JE-3, KC-1, and ornithine decarboxylase), and an S-phase gene (histone H3). The expression of these growth-regulated genes was studied in both tsJT60 cells and its parental cell line, rat 3Y1 cells. All the genes tested, except histone H3, are similarly induced when quiescent tsJT60 cells are stimulated by serum at either permissive or restrictive temperatures. These results raise intriguing questions on the nature of quiescence and the relationship between G0 and G1 in cells in culture.     

A temperature-sensitive cell-cycle mutant of mammalian cells, tsJT16, is defective in a function operating soon after growth stimulation

A temperature-sensitive cell-cycle mutant, tsJT16, which has been isolated from Fischer rat fibroblasts, was defective in the function(s) that operated soon after growth stimulation. When G0-arrested tsJT16 was stimulated to proliferate, it entered the S phase after 12-15 h at 34 degrees C but failed to do so at 40 degrees C. The function mutated in tsJT16 was required to be normal for the first 4 h or less for cells to transit from the G0 to S phase. The induction of cell-cycle-dependent genes such as c-fos, c-myc and ornithine decarboxylase was observed at both temperatures after growth stimulation. Although an increase in total protein synthesis occurred at both temperatures after growth stimulation, synthesis of one protein (p70) (pI 7.8 and Mr 70,000) was inhibited at 40 degrees C. Synthesis of p70 was negligible in G0-arrested cells and blocked by actinomycin D in serum-stimulated cells at 34 degrees C. These results suggest that tsJT16 has a ts defect in one of the signal transduction processes to induce gene activation. tsJT16 was also defective in progression of the G1 phase of growing cells, consistent with the previous results in which growth stimuli were required at G1 for continuation of proliferation.     

A nuclear labile protein, p70, is expressed exclusively during G0-S transition but not in G1 phase of a cell cycle ts mutant, tsJT16

tsJT16 is a cell cycle temperature-sensitive (ts) mutant from a Fischer rat cell line. When it is growth-stimulated from G0 phase it enters S phase at the permissive temperature (34 degrees C) but not at the nonpermissive temperature (40 degrees C). It induces a nuclear labile protein, p70, when it is stimulated from G0 phase at 34 degrees C, but not at 40 degrees C. In growing cell cycle it progresses through the S, G2 and M phases at both temperatures but fails to pass through G1 phase at 40 degrees C. Here we described that p70 was synthesized neither in the randomly growing cycle nor in the G1 phase synchronously progressing from M phase. The cells synchronized at early G1 phase by culturing in serum-free medium for 7.5 h from G1/S boundary induced c-fos and c-myc following serum addition, but under the same condition p70 was not synthesized. These results indicate that the synthesis of p70 is not required for progression of the G1 phase of the growing cycle and can be used as an exclusive marker of G0-S transition.     

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.     

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.     

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.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

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.     

Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.     

Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts

Four temperature-sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8 degrees C are shifted up to 39.8 degrees C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8 degrees C. We studied the traverse through the S and G2 phases at 39.8 degrees C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8 degrees C were shifted down to 33.8 degrees C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.     

Prolongation of duration of G2 arrest delays and finally blocks entry into M phase in contrast to stable and reversible G1 arrest: study of a G1/G2 temperature-sensitive mutant of rat 3Y1 fibroblasts

Proliferation of 3Y1tsF121 cells was arrested in G1 and G2 phases after a shift up to 39.8 degrees C (restrictive temperature). Both arrests were reversible: after a shift down to 33.8 degrees C (permissive temperature), these cells effectively entered the next phases. However, the entry into M phase of the G2-arrested cells was delayed depending on the time in arrest. The G2-arrested cells finally became incapable of entering M phase with a prolonged incubation at 39.8 degrees C. Under the same condition, G1-arrested cells did not lose their ability to proliferate, and their delay of entry into S phase was slight. Therefore, cells in G2 phase are, in a sense, more unstable than the cells in G1 phase. These results also suggest that the time required for entry into M phase may depend on the preparedness for the initiation of M phase and, that it may be prolonged under the condition where the preparedness for entry into M phase is diminished.