Use of low temperature for growth arrest and synchronization of human diploid fibroblasts

The growth kinetics of human diploid fibroblasts at two different temperatures were followed. Proliferation of exponentially growing cells is reduced and eventually stops upon incubation at low temperature (i.e. 30°C). The cells which are in S phase at the time of switching to low temperature complete their DNA synthesis and become arrested in the G₁ phase of the cell cycle. The arrested cells can be stimulated to proliferate by restoration of the optimal growth temperature (37°C). The kinetics of entry into S phase were investigated by measuring [³H]thymidine incorporation into TCA-precipitable material, by autoradiography and by flow cytofluorimetry. The synchronized cells initiate DNA synthesis at approximately 8 h and DNA synthesis peaks at 20.4±0.7 h after stimulation.In addition, the rates of UV-induced excision repair at 30°C and 37°C were compared. The results indicate that at 30°C the excision-repair process is operative but at a slightly reduced rate in comparison with repair at 37°C.This method will be useful for the study of S-phase-dependent processes, as well as for repair studies in the absence of cell division.     

Effect of temperature on growth and macromolecular biosynthesis in cryptococcus species

Cryptococcus neoformans, a pathogenic yeast, grows at temperatures between 25 and 37°C. However, the closely related non-pathogen C. albidus exhibits restricted growth at temperatures above ambient with little or no growth at 37°C. The inhibition of growth of the non-pathogen, as measured by turbidity, cell number, and per cent budding, is reversible after 48 hr at the non-permissive temperature (37°C). Growth cessation at 37°C is accompanied by a corresponding decrease in DNA synthesis, which is not observed in C. neoformans. RNA and protein synthesis in C. albidus and C. neoformans are only slightly affected at the elevated temperature. Degradation by nucleases does not seem to account for the differences found in this cumulative DNA synthesis in C. albidus at 25 and 37°C. These facts suggest that C. albidus may possess a thermo-sensitive defect in the machinery responsible for the initiation of DNA replication.     

Sea urchin egg fertilization studied with a fluorescent probe (ANS)

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10^?14 g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Thermolabile analogue proteins and the entry of HeLa and CHO cells into division

Entry of HeLa and CHO-10 cells into mitosis can be inhibited by incorporation of p-fluorophenylalanine at certain temperatures, 37 °C for the former cell type and 39.5 °C for the latter. At lower temperatures, 32 °C in the former and 37 °C in the latter, the analogue does not inhibit entry of cells into mitosis. The possibility that the analogue is not incorporated at the permissive temperatures has been ruled out; indeed incorporation is relatively greater at the permissive temperatures. The results suggest that the physiological properties of analogue protein molecules differ depending on the temperature at which they are synthesized; the higher the temperature the more likely they are to malfunction.     

Phospholipid metabolism in Mycobacterium smegmatis ATCC 607 grown at 37° C and 27° C

The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of ³²P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine (PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids at the two temperatures have been discussed.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1.

AF8, a temperature-sensitive mutant of BHK $\text{21}\text{13}$ cells, has been shown to arrest at the non-permissive temperature in the G1 phase of the cell cycle. When AF8 cells are released from density-dependent arrest of growth by trypsinization and replating at lower density, 60–80% of the cells enter DNA synthesis and divide at the permissive temperature (33 °C), while only 20% enter DNA synthesis at the non-permissive temperature (39.5 °C). The temperature-sensitive block has been localized 4 to 8 h before the onset of DNA synthesis which begins at 12 h after stimulation. Two biochemical events of the prereplicative phase have been temporally related to this temperature-sensitive block. RNA synthesis as measured in isolated nuclei increases initially at both temperatures, then levels off and declines to control levels at 39.5 °C while continuing to increase at 33 °C. Parallel changes are found in circular dichroism spectra and ethidium bromide binding capacity of isolated chromatin. The results suggest that these biochemical changes are involved in the regulation of the prereplicative phase and the subsequent entrance of cells into DNA synthesis.     

Thermally Enhanced Radiosensitivity of Cultured Chinese Hamster Cells

X-IRRADIATION of mammalian cells in culture yields a survival curve of the threshold type (for review see ref. 1). It isjnter-esting to ask how one can enhance the radiation response by small changes of the physical environment of the cells, as can be done chemically, for example, by incorporation of 5-bromo-deoxyuridine into DNA^1,2. Elevation of the temperature is a likely prospect for enhancement of radiosensitivity for the following reasons. It is known that proteins are heat labile and that temperature sensitive mutants of bacteria and phage can be obtained for many different enzymes³ which are operative at 37° C but not at 42° or 43°C. For example⁴, DNA polymerase is reversibly temperature sensitive; it is rendered inoperative above 42°C, but will be functional again when the temperature is lowered. It is not unreasonable to expect that temperature sensitive mutations for many enzymes occur frequently and that the use of temperatures somewhat higher than the normal range at which the cells grow might disclose sensitivities for specific enzymes in normal cells of higher organisms.     

Pyrimidine Deoxyribonucleosides Are Phosphorylated to Triphosphates at Low Temperatures Too,But Are Not Incorporated into DNA or Phospholipids.

Abstract

Thymidine (Thd) was phosphorylated to dTTP also at 0°C, both in Ehrlich ascites tumor cells and human tonsillar lymphocytes, but was not incorporated into DNA. The uptake and phosphorylation of ¹⁴C-Thd into the pool showed regular kinetics (Km 6, 6 uM), and the main metabolite was dTTP (75–84%) both at 0°C, and 37°C. Similarly, deoxycytidine (dCyd) was also transported and phosphorylated to nucleotides (76%) at low temperature, but no incorporation into DNA and phospholipid precursor liponucleotides could be detected at 0°C. Under the same conditions, at 37°C, when lymphocytes were labeled with 5-³H-dCyd, 51% of the total pool radioactivity was found in liponucleotides. Transport and phosphorylation of deoxynucleosides seem to be tightly coordinated at both temperatures, which processes are directly coupled to membrane-phospholipid and DNA biosynthesis, but only at physiological temperature while they seem “uncoupled” at low temperature. The fact that nucleoside phosphorylation occures also at low temperature has implications for several experimental techniques used in cell biology.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

Ploidy evolution in the yeast Saccharomyces cerevisiae: a test of the nutrient limitation hypothesis

The nutrient limitation hypothesis provides a nongenetic explanation for the evolution of life cycles that retain both haploid and diploid phases: differences in nutrient requirements and uptake allow haploids to override the potential genetic advantages provided by diploidy under certain nutrient limiting conditions. The relative fitness of an isogenic series of haploid, diploid and tetraploid yeast cells (Saccharomyces cerevisiae), which were also equivalent at the mating type locus, was measured. Fitness was measured both by growth rate against a common competitor and by intrinsic growth rate in isolated cultures, under four environmental conditions: (1) rich medium (YPD) at the preferred growth temperature (30 °C); (2) nutrient poor medium (MM) at 30 °C; (3) YPD at a nonpreferred temperature (37 °C); and (4) MM at 37 °C. In contrast to the predictions of the nutrient limitation hypothesis, haploids grew significantly faster than diploids under nutrient rich conditions, but there were no apparent differences between them when fitness was determined by relative competitive ability. In addition, temperature affected the relative growth of haploids and diploids, with haploids growing proportionately faster at higher temperatures. Tetraploids performed very poorly under all conditions compared. Cell geometric parameters were not consistent predictors of fitness under the conditions measured.     

The initiation and elongation steps in protein synthesis: Relative rates in chinese hamster ovary cells during and after hyperthermic and hypothermic shocks

The relative rates of the initiation and elongation phases of protein synthesis have been determined in heat- and cold-shocked CHO cells from measurements of the incorporation of ³⁵S-methionine into N-terminal and internal positions of growing peptides by a modified Edman degradation. When the cells are shifted from 37°C to temperatures between 10°C and 34°C, the rate of initiation is at first reduced more extensively than that of elongation. After 20 to 30 minutes at the lower temperature, however, the cells undergo a metabolic adjustment which includes increasing the rate of initiation until it corresponds to the rate of elongation at that temperature. Calculated apparent energies of activation for initiation and elongation are in reasonable agreement with those determined in other mammalian cells. When the cooled cells are returned to 37°C, the rates of initiation and elongation recover immediately but do not exceed the control values. Exposure to elevated temperature (43°C) causes an immediate cessation of initiation and thus a delayed inhibition of elongation; upon return to 37°C, the rate of initiation is transiently elevated above the control rate, and the rate of elongation returns to the control rate after a 2- to 3-minute delay. Hence, a factor which leads to supranormal rates of initiation may accumulate at high but not at low temperatures.     

Maintaining mosquito cell lines at high tempetatures: Effects on the replication of flaviviruses

Summary The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32°C to 34°C,Anopheles stephensi cell line could be maintained at 37°C. At higher temperatures initial growth rate was higher, but yield of cells after about a week of incubation was lower than at the standard temperature (28°C). Replication of several flaviviruses inAedes albopictus cell cultures adapted to 34.5°C was faster, and viral titers were higher than at 28°C.     

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [3H]-flunitrazepam binding in rat brain

The competitive inhibition of [³H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Nontemplate Polymerization of Free Nucleotides Into Genetic Elements by Thermophilic DNA Polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life.

Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides, and Nucleic Acids to view the free supplemental file.     

Regulation of DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus.

The onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus-transformed derivatives after a period of serum deprivation. Rat-1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non-permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non-permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non-permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non-permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat-1 cells and these cells transformed by wild-type RSV (Rat-1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum-dependent process during G₁ necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum-related control mechanisms ineffective in the regulation of growth.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.