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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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 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.     

The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.     

Cytoplasmic regulation of two G1-specific temperature-sensitive functions

tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state.     

Advance toward S phase and retreat toward deeper "G0" states in resting 3Y1 cells with environmental changes

To elucidate conditions which affect the lag time for resting cells to enter S phase after serum stimulation, we used a wild-type 3Y1 rat fibroblast line and four temperature-sensitive mutants of 3Y1 (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203). Among these five lines, in only tsG125 cells was there an obviously prolonged lag time with increase in time in resting state at 33.8 degrees C. The resting wild-type 3Y1 cells, preexposed to 39.8 degrees C, also showed a prolongation of lag time. The prolongation in tsG125 had a certain limit. Preexposure to 39.8 degrees C before serum stimulation accelerated such prolongation in tsG125 to its limit, but did not change the limit, per se. Resting tsG125 cells stimulated by serum at 39.8 degrees C, did not enter S phase, yet they did advance toward S phase. When they were kept at 39.8 degrees C, they retreated toward a deeper resting state ("G0") with time. These retreats correlated with the decrease in stimulating activity in the culture media. About 20% of the resting tsG125 cells stimulated by serum at 39.8 degrees C were committed to enter S phase, when the extent of commitment was examined at 33.8 degrees C. Most of the tsG125 cells committed at 33.8 degrees C did not enter S phase, when the extent of commitment was examined at 39.8 degrees C. More cells were committed after stimulation at 33.8 degrees C than at 39.8 degrees C, when the test was done at 33.8 degrees C. We suggest that resting cells may be reversibly changed within range of resting states, in either direction, that is, advance toward S phase or retreat toward deeper "G0." These changes may be determined by alterations in the balance between synthesis and decay of the preparedness for the initiation of DNA synthesis caused by cellular response to environmental changes (e.g., medium activity, temperature, etc.). The ts defect in tsG125 may affect the cell cycle progression, both before and after commitment by serum.     

Expression of histone genes in a G1-specific temperature-sensitive mutant of the cell cycle

The expression of genes coding for the four core histones (H2A, H2B, H3, and H4) was studied in tsAF8 cells. These baby hamster kidney-derived cells are a temperature-sensitive (ts) mutant of the cell cycle that arrest in G1 at the restrictive temperature. When serum-deprived tsAF8 cells are stimulated with serum, they enter the S phase at the permissive temperature of 34 degrees C, but are blocked in G1 at the nonpermissive temperature of 39.6 degrees C. Northern blot analysis using cloned human histone DNA probes detected only very low levels of histone RNA either in quiescent tsAF8 cells or in cells serum stimulated at the nonpermissive temperature for 24 h. Cellular levels of histone RNA were markedly increased in cells serum stimulated at 34 degrees C for 24 h. Temperature shift-up experiments after serum stimulation of quiescent populations showed that the amount of histone RNA was related to the number of cells that entered the S phase. Those cells that synthesized histone RNA and entered the S phase were capable of dividing. This is the first demonstration in a mammalian G1-specific ts mutant that the expression of H2A, H2B, H3, and H4 histone genes depends on the entry of cells into the S phase of the cell cycle.     

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.