Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle.

C W Gibson,R R Hirschhorn,W E Mercer,S Rittling,H T Liu,R Baserga

doi:10.1016/s0021-9258(19)83616-5

C W Gibson, R R Hirschhorn + Show 4 more

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https://doi.org/10.1016/s0021-9258(19)83616-5

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Abstract

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.

Highlights

The levels of thymidine 2A9, two cDNA clones whose sequences are expressed in a kinase mRNA andenzymeactivity increased markedly cell cycle-regulated manner, is increased in serum-stimulated in both cell lines serum stimulated from quiescence at ts13 cells at both pt’ and npt [10]
When ts13 cells are serum stimulated at the nonpermissivetemperaturedihydrofolatereductase enzyme activity declines rapidly and dihydrofolate re- CelIs-The cell lines used, tsAF8 and ts13 cells, are described in ductase mRNA is below detectablelevels
Levels ofDihydrofolate ReductasemRNA after Serum Stim- The secondcontrol gene isa cDNA clone,p2A9; expression ulation-Thelevels of dihydrofolate reductase mRNA of sequences corresponding to the insert ofp2A9 markedly increase after quiescent ts13 and tsAF8 cells are serum stim- increaseswithin 16 h after quiescent ts13 cells are stimulated ulated at pt;,in tsAF8they are alreadyat a detectable level in quiescent cells(Fig. 6)

Summary

RESULTS

Our results can be better understood if we first summarize the characteristics of the two cell lines we used, ts and tsAF8 cells. At pt serumstimulated ts cells enter S phase with a median lag time of 25 h, and theexecution point of the ts mutation is -3 h before S or 22 h after serum stimulation [25]. Thymidine Kinase Activity after Serum Stimuhtwn"tsl and tsAF8 cells were made quiescent by serum deprivation and subsequently stimulated with 10% serum at either pt or npt. Thymidine kinase activity remains high at pt even 48 hafter serum stimulation. It should be noted, that under the conditions of our experiments the cells go through more than one cell cycle.At

HOURS AFTER SERUM STIMULATION

HOURS AFTER SERUM

DISCUSSION

HOURS A F T E RS E R U M