Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

Abstract

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39°C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.In procaryotic organisms, conditional lethal mutations affecting DNA replication have been useful in identifying proteins essential to the synthesis of DNA and its precursors (21) Mutations that specifically affect the synthesis of deoxyribonucleoside triphosphates (dNTPs) have helped to clarify metabolic and genetic relationships between deoxyribonucleotide synthesis and DNA replication (41, 42). For example, mutations in the genomes of both Escherichia coli and bacteriophage T4 have been used to generate evidence supporting a direct physical connection between the replication apparatus and the enzymatic machinery for dNTP synthesis (29). No such temperature-sensitive mutants have been described, however, for mammalian cells. Consequently, we investigated the nature of the biochemical defect in JB3-B. We report here evidence demonstrating that a defect in dNTP synthesis is responsible for the temperaturesensitive phenotype in JB3-B. Infected mutant cells exhibited a temperature-dependent defect in Ad DNA synthesis. There was also a depletion of dNTP but not ribonucleoside triphosphate (rNTP) pools when uninfected cells were shifted from 33 to 39°C. Furthermore, the mutant cells were found to contain a thermolabile form of ribonucleotide reductase activity. Unexpectedly, the changes in dNTP pool size that resulted from shifting a culture of JB3-B to a nonpermissive temperature (NPT) was quite distinct from those ...

Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation

“…In mutant fibroblasts lacking dCMP deaminase there is a decreased dTTP pool whereas dCTP accumulates [29] ; there could therefore be a decrease in deaminase activity in IL-3-deprived cells. In contrast with the BAF3 cell model, where the cells are either growing or undergoing apoptosis, there are situations where quiescence is the alternative to growth, such as temperature shifting of thermolabile CHO mutant cells [30] or activation of human peripheral blood lymphocytes by phytohaemagglutinin [31,32] ; in these cases the resulting perturbations in dNTP levels show a co-ordinated change with no imbalance of the DNA precursors. dNTP pool balance has been shown to be essential for fidelity in DNA synthesis in dividing cells (reviewed in [33]), and inhibition of DNA synthesis is sufficient to cause cell death [15].…”

Evidence for immortality and autonomy in animal cancer models is often not provided, which causes confusion on key issues of cancer biology

“…There are even transgenic animals established to facilitate the establishment of such conditionally immortalized cell lines [755]. Some of these conditionally immortalized cells can form colonies in soft agar when the coercer is turned on, but no colony is formed when it is turned off [756,759,761]. Obviously, the "conditional" means reversable, implying that the immortality or transformation occurs simply under the duress of the immortalizer or the transforming gene, and not due to the relevant epigenetic or genetic alterations.…”

Mutation or not, what directly establishes a neoplastic state, namely cellular immortality and autonomy, still remains unknown and should be prioritized in our research

“…However, for different research needs, many systems of "conditional immortality" or "conditional transformation" have also been created [600][601][602][603][604][605], including transgenic animals [606]. Accordingly, many conditional cell lines have been established [589,[606][607][608][609][610][611][612][613][614][615], like the temperaturecontrolled ones [612,616], which show controllable immortalization or neoplastic transformation [612,615,617]. The words "conditional" and "controllable" already proclaim the nature of swift reversibility and accentuate that the immortality or the neoplastic transformation so created is not authentic because the cells are still mortal.…”