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.     

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.     

Ribosome biosynthesis is not necessary for initiation of DNA replication.

Synthesis of mature 28-S ribosomal RNA and 60-S ribosomal subunits is inhibited in baby hamster kidney (BHK) cell line ts 422E at non-permissive temperature (39 degrees C). This leads to a 66% decrease of total ribosomes per cell, a marked imbalance between the large and small ribosomal subunits in the cytoplasm and a decrease of cells per dish after prolonged culture at 30 degrees C. However, inhibition of ribosome synthesis does not affect progression of cells through the G1 period of the cell division cycle, the length of the pre-replicative period, and the rate of entry of cells into S phase. In contrast to culture at non-permissive temperature, culture of BHK ts 422E cells in the presence of 0.04 micrograms/ml actinomycin D at 33 degrees C inhibits markedly the entry into S period. It is concluded that low doses of actinomycin D exert their inhibitory effect on cell growth by preventing maturation and transport of mRNA rather than by interfering with ribosome synthesis. Microfluorometric analysis revealed only slight differences in the distribution of BHK ts 422E cells in G1, S and G2 phases of the cycle either when cultured at 33 degrees C or at 39 degrees C. When too few ribosomes per cell are produced in BHK ts 422E cells at 39 degrees C, cells do not seem to be arrested reversibly at a specific point of the cell cycle but rather to die at random.     

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. III. Large T antigen level and cell proliferation and survival in an SV40 tsA640-transformed macrophage line

The levels of simian virus 40 (SV40) large T antigen in a tsA-transformed mouse macrophage line at the permissive (33 degrees C) and the nonpermissive (39 degrees C) temperature were examined by immunofluorescence, sodium dodecylsulfate-polyacrylamide gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. When the cells were confluent and rested at 33 degrees C, and then were shifted to 39 degrees C, the amount of large T antigen per cell decreased, and most cells survived and remained phagocytic. When the cells were proliferating at 33 degrees C, and then were shifted to 39 degrees C, the cells died with only a small reduction in the amount of large T antigen. Therefore, the physiological state of the cells may determine the survival of cells by affecting the level of large T antigen after exposure to 39 degrees. The confluent cells may be rested with a concomitant decrease of large T antigen. The proliferating cells may not survive in the presence of a relatively high level of functionally defective large T antigen at 39 degrees C.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.     

Rate of progress of the 1st post-stimulation division of the mitotic cycle by cells of ascitic hepatoma 22A of different ages]

A study was made of the progress rate of cells of the ascitic hepatoma 22A of different age during the iirst mitotic cycle after the stimulation of division. The "ageing" (11-day), terminal (14-day), and "delayed" (4 days older than the terminal stage) ascitic fluids were used. The maximal values of the labeled nuclei index was found to be reached by 9--12 hours (it was mainly due to the transtion of the quiescent to the S-period) and the maximal mitotic index--by 18--21 hours after the inoculation, independently of the tumour age. These results suggest that the duration of both the prereplicative (G1) period and of the whole first mitotic cycle after the stimulation were independent of the time during which the cells of the ascitic hepatoma 22A were at the resting stage or at the very prolonged G1-period.     

Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells.

Proliferation of mammalian cells can be controlled by low cultivation temperature. However, depending on cell type and expression system, varying effects of a temperature shift on heterologous protein production have been reported. Here, we characterize growth behavior and productivity of the Chinese hamster ovary (CHO) cell line XM111-10 engineered to synthesize the model-product-secreted alkaline phosphatase (SEAP). Shift of cultivation temperature from 37 degrees C to 30 degrees C caused a growth arrest mainly in the G1 phase of the cell cycle concomitant with an up to 1.7-fold increase of specific productivity. A low temperature cultivation provided 3.4 times higher overall product yield compared to a standard cultivation at 37 degrees C. The cellular and molecular mechanisms underlying the effects of low temperature on growth and productivity of mammalian cells are poorly understood. Separation of total protein extracts by two-dimensional gel electrophoresis showed altered expression levels of CHO-K1 proteins after decrease in cultivation temperature to 30 degrees C. These changes in the proteome suggest that mammalian cells respond actively to low temperature by synthesizing specific cold-inducible proteins. In addition, we provide the first evidence that the cold response of mammalian cells includes changes in postranslational protein modifications. Two CHO proteins were found to be phosphorylated at tyrosine residues following downshift of cultivation temperature to 30 degrees C. Elucidating cellular events during cold exposure is necessary for further optimization of host-cell lines and expression systems and can provide new strategies for metabolic engineering.     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Indices of mitotic cell division in sebaceous glands]

The main indices of mitotic cell division in rat sebaceous glands (external auditory meatus and tarsales gl.) were studied autoradiographically using H3-thymidine and with colchicine method. The duration of mitotic cycle and its separate phases, the number of cells involved in the proliferative pool, as well as the turnover of terminals of the epithelium in both the glands were stated to be nearly identical. The duration of the mitotic cycle was: T -- 28.1 hour; tG1 -- 18.64; tS -- 6.3; tG2 -- 1.80; tM -- 1.34 hours. The proliferative pool (Pc) -- 31.45%, turnover of the basal layer cells -- 89.25 hours. These indices for the stratified epithelium of excretory ducts were respectively; T -- 33.0 hours; tG1 -- 21.74; --8.06; tG2 -- 1.6; tM -- 1.6; Pc -- 26.8% and the turnover for the cells of the basal layer -- 123 hours. Thus, the sebaceous glands are to be regarded as organs where a rapid renovation of epithelia cells occurs.     

Viscoelastic and light scattering studies on thermally induced sol to gel phase transition in fish myosin solutions

Viscoelastic (VE) and dynamic light scattering (DLS) analyses of fish (white croaker) myosin solutions were performed at myosin concentrations of 30 mg/mL for VE and 0.1 mg/mL for DLS at 0.6M KCl and pH 7.0 to clarify thermally induced gelation. The hydrodynamic radius R(h) considerably decreased around 30-35 degrees C. The shear modulus G was constant below 25 degrees C and increased by incubating the sample at 30 degrees C. G further increased as the temperature of the incubated sample decreased. The curves of G vs T for different time courses showed a sharp peak around 35 degrees C and a moderate peak around 60 degrees C in the heating process, while a stepwise increase in G was observed around 30 degrees C in the cooling process when the temperature was elevated to not more than 60 degrees C. No distinct stepwise change was observed once the temperature of the sample exceeded 60 degrees C. The absolute value of G strongly depended on the maximum elevated temperature and the incubation time at that temperature. The corresponding behavior of the viscosity eta was observed for each time course. Based on these results, the mechanism of thermally induced gelation of myosin solutions is discussed in view of S-S bridge formation in the head and tail portions and unwinding/rewinding of coiled-coil alpha-helices in the tail portion.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.     

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.     

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.     

Effect of serum on heat response of synchronized mouse neuroblastoma cells: protection of cell cycle progression, protein synthesis and survival

The effect of serum and temperature elevation on proliferation has been studied in synchronized mouse neuroblastoma (Neuro-2A) cells. The effects of serum were studied on the induction of (a) mitotic delay due to a non-lethal heat treatment (30 min at 42.7 degrees C) and (b) the loss of colony-forming capacity after a more extensive heat treatment (45 min at 44 degrees C or a continuous 42.7 degrees C heat treatment). The following results were obtained. Under conditions of serum depletion, cell cycle extension of heated G1 phase cells was more than that of heated G2 phase cells. Serum protected against heat-induced alterations of cell cycle progression in G1- but not in G2 phase cells. This effect of serum could be mimicked by a supplement to the medium of human transferrin, bovine pancreas insulin and selenium, and was correlated with protection of protein synthesis. Serum also affected heat-induced cell killing. Under conditions of serum depletion, G1 phase cells were more resistant to heat compared to G2 cells. The presence of serum during heat treatment further increased the thermoresistance of G1 phase cells, but did not affect sensitivity of G2 phase cells. This effect of serum could not be mimicked by a supplement of transferrin, insulin and selenium. These results indicate that serum protects G1 phase cells for heat-induced changes of cell cycle progression as well as on cell survival, but the mechanisms involved in both phenomena seem to be different.     

The effect of ultrahigh frequency electromagnetic fields on the reduction of ferricyanide by human erythrocytes in the presence of methylene blue

Effect of microwave radiation with the frequency of 1000 +/- 10 MHz and specific absorption rate of 220-580 mV/g on the ferricyanide reduction by human red blood cells in the presence of methylene blue (carrier of oxidation-reduction equivalents through the membrane) was studied at different temperatures in the region of 23-34 degrees C. The temperature dependence of the ferricyanide reduction rate in Arrhenius plots shows two sharp "anomalous" sites with apparently negative activation energy at 26-27 and 29-30 degrees C. Broadness and expression of the "anomalous" sites increased with an increase of the blood storage time. The increase of the ferricyanide reduction rate under microwave irradiation was observed only in the temperature regions corresponding to the "anomalous" sites of the temperature dependence.     

Experiments with cultures of mammalian cells aboard the biosatellite "Cosmos-782"

A considerable contribution to the investigation on biological importance of weightlessness was made by the experiments with animals in the artificial Earth satelites (AES) of "Cosmos" type. Cell cultures can serve as an ideal model to get a direct cell response to the effect of external factors. For the experiment in the AES "Cosmos-782", two thoroughly examined cell strains (L and 237) were chosen, which differed in a number of parameters (for example, duration of their mitotic cycles). Density of cell seeding and temperature of their cultivation in the laboratory experiment were calculated in such a way that the whole cycle of the culture development should take place under the conditions of weightlessness: the beginning of lag-phase--before launching and the stationary phase--after landing. The weightlessness was not shown to result in any genetical shifts revealed at chromosomal level. When cultivated after the flight, the cells do not change their mitotic cycle parameters, mitotic course and structural organization. The data obtained in the experiments with AES "Cosmos-368" and "Cosmos-782" (increase of mitotic index, some forms of mitotic pathology during the first terms of cultivation after the flight and enlargement of cellular nuclei) demonstrate the changes in the cell population which have formed under the conditions of weightlessness. Similar changes are observed while the cells propagate in the laboratory conditions. Indirect data on an earlier cell culture aging during the flight do not exclued the possibility that under weightlessness the rate of cell propagation could differ from that under gravitation.     

Relative competitiveness of haploid and diploid yeast cells growing in a mixed population

Saccharomyces cerevisiae was grown in a rich medium under the conditions of "quasi-continuous" cultivation and, after 200-300 generations, its diploid cells almost completely displaced haploid cells from the original mixed "haploid-diploid" population where the ratio between diploid and haploid strains was either 1:1 or 1:100. The cultivation at 40 degrees C did not change the relative competitive ability of haploids and diploids. When cells were cultivated in a rich medium at 6 degrees C or in a minimal medium at 30 degrees C, none of the strains showed an advantage over others for about 200 generations. Haploid cells had an advantage over diploid cells during "quasi-continuous" growth in the minimal medium at 30 degrees C. When the temperature was elevated to 40 degrees C, diploid cells displaced haploid cells from the mixed population. No advantage was found for diploid or haploid cells grown in a medium with an elevated KCl content (1.5 M). Haploid cells had an advantage over diploid cells when Pichia pinus was cultivated in a minimal medium. The results are discussed using the hypothesis about the diploid phase being fixed in the course of biological evolution.     

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.     

The sequence and duration of mitotic cycles in the apical root meristem ofVicia faba l

A study was made on the mitotic cycle times in meristematic cells of Vicia faba root tips and on the relationship between their duration and the position of a certain cell in the column of proliferating cells. For the demonstration of the sequence and duration of mitotic oycles a single-column model was used. The results of experiments show that the great variability in the duration of mitotic cycles (from 12 to l20 h)is the result of a different program of the apical meristem cells. The long duration of the cycle of initials corresponds to the sum of cycle times of their descendants.