DNA synthesis and mitosis in a temperature sensitive Chinese hamster cell line

Abstract

Viability, DNA synthesis and mitosis have been followed in the temperature sensitive Chinese hamster cell mutant K12 under permissive and non-permissive conditions. On incubation a t 40°C cells retained their ability to form colonies at 3 3°C for 15 to 20 hours, but viability was lost gradually during the following 20 hours. When random cultures of K12 were shifted to 40°C the rate of DNA synthesis was normal for three to four hours but then decreased markedly, reaching 95% inhibition after 24 hours. Under the same conditions mitosis was inhibited after 15 hours. If cultures which had been incubated at 40°C for 16 hours were placed at 33°C the rate of DNA synthesis increased five hours after the shift down and mitosis 18 hours after. These results can be interpreted on the assumption that K12 at 40°C is unable to complete a step in the cell cycle which is essential for DNA synthesis and which occurs three to four hours before the start of S at 33°C.The preceding paper described the isolation of a temperature sensitive mutant K12 derived from the Chinese hamster cell line Wg-1A (Roscoe, Read and Robinson, '73). K12 was selected from a large population because it detached relatively easily from a glass substrate at the nonpermissive temperature. It was found to have a very low spontaneous reversion rate (less than 1 in 6 X lo7) and over a period of 48 hours at 40°C K12 cells stopped growing and became fully rounded in contrast to a normal spread configuration and good growth at 33°C. In this paper experiments with K12 and Wg-1A are reported which describe the effect of temperature shifts between 33 O C and 40 O C on viability, DNA synthesis and mitosis. MATERIALS AND METHODS CellsWg-lA, the isolation of K12 and the growth medium used have been described previously (Roscoe, Read and Robinson, '73). Stocks of cells were maintained at 31 "C with transfers at weekly intervals. Experiments were carried out at 33°C or 40°C in plastic bottles (Falcon Plastics) or Petri dishes (NUNCLON).For continuous observation of cells at 33°C or 40°C micrographs of cultures were taken at intervals of 90 or 120 seconds using 16 mm cine film. When cells were shifted from low to high temperature or vice-versa the constant temperature cabinet and microscope equilibrated within one hour. The resulting films were analysed using a cine projector fitted with a frame counter. Measurement of D N A synthesis: (i) By thymidine incorporationThe method used was based on that described by Bachetti and Whitmore ('69). All the Petri dishes (50 mm diameter) for a given experiment were inoculated from one cell suspension with about 5 X 104 cells; they were then incubated for 24 hours at 3 3°C before a n y required temperature shifts. In these experiments operations at 40°C were performed in a constant temperature room. For each point at which the rate of DNA synthesis was to be measured the medium was carefully removed and replaced with fresh growth medium containing 0.5 pC/ml "H-thymidine (TdR; 5.25 C per mmole). In some experiments "H-TdR was a...

Use of a cell cycle mutant to delineate the critical period for the control of histone mRNA levels in the mammalian cell cycle.

“…The hamster mutant cell line K12 used in this study was originally isolated from an established line of Chinese hamster fibroblasts, Wg1A, after treatment of the cells with ethyl methane sulfonate (30,31). K12 was selected because it grows normally at 35°C but is arrested in Gl upon incubation at 40.5°C.…”

Testicular Feminization Associated with a Thermolabile Androgen Receptor in Cultured Human Fibroblasts

“…The amount of binding in control cell strains changed little when the assay temperature was increased to 42°C; some 3r 42e 37 The phenomenon of temperature sensitivity as an indication of a structural mutation in a protein was first noted in bacterial mutants in which elevation of temperature is associated with altered growth (19). More recently, mutations have been characterized in mammalian cells in which elevated temperature inhibits the normal cell cycle (20,21). In some ofthese systems a temperature-sensitive protein, usually an enzyme critical for normal cell growth and division, has been found to be structurally abnormal and responsible for the effects of elevated temperature on cell growth (22)(23)(24).…”

Isolation of a Temperature-Sensitive Cell Cycle Variant of Chinese Hamster Cells

“…Roscoe et al (1973) and Smith and Wigglesworth (1973) reported that in a variant K12 of Chinese hamster cells, the temperature-sensitive lesion is in the late G1 phase. Burstin et al (1974) found that the arrest point of a variant tsAE8 of BHK cells is located in the G1 phase between the blocks induced by serum starvation and isoleucine deprivation.…”