John Morrow

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

Abstract The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.

Regulation of Synthesis of Amyloid A-Related Protein*

The concentration of serum amyloid A polypeptide (SAAL) increases greatly during the acute phase responses to infection or inflammation. We find that SAAL synthesis comprises 2.5% of murine hepatic protein synthesis after lipopolysaccharide (LPS) administration, but much less in normal liver. SAAL messenger RNA (mRNA) in liver increases at least 500-fold above the normal level. A recombinant plasmid homologous to SAAL mRNA has been isolated, as has most of the mouse genome DNA encoding the plasmids nucleotide sequence. This gene is transcribed into RNA much more frequently after LPS administration than it is in normal liver. In a number of other mammalian genes, cytosine methylation is inversely related to the rate of transcription. Methylation of CCGG sequences in hepatic DNA homologous to the recombinant plasmid has been examined. Little or no change is found after LPS administration. This suggests that other factors are responsible for the increase in SAAL mRNA in the acute phase response.

Gene inactivation as a mechanism for the generation of variability in somatic cells cultivated in vitro

Abstract It is proposed that a substantial proportion of cell culture variants result from repression or derepression of genetic information which specifies the synthesis of enzymes. The mechanism responsible for this process may be similar to the inactivation of euchromatic segments in Drosophila resulting from their transposition next to heterochromatin. This model could explain a number of confusing observations made in somatic cell culture systems. (1) The generally high mutation rate for markers in somatic cells. (2) The instability and high reversion rates of a number of genetic markers. (3) Failure of certain drug resistance markers to show the expected decreases in mutation rate with increase in ploidy levels. (4) Anomalous mutation induction kinetics, and lack of specificity of mutagenic agents. Means by which this model can be experimentally examined are proposed. They include: (a) correlation of late labeling patterns and transposition of genetic material with phenotypic changes suspected of having their basis in chromosomal inactivation. (b) Mutagenesis studies using markers which would be expected to show phenotypic variation due to chromosomal inactivation. (c) Studies of gene expression in somatic cell hybrids. (d) Examination of gene products from resistant cell lines suspected of arising from chromosomal inactivation.

An Evaluation of Some Theories of the Mechanism of Aging

Two theories of aging are considered in this review. Although there exists substantial experimental evidence in support of the somatic mutation and error catastrophe hypotheses, several experiments have been published which are extremely difficult to reconcile with these models, at least in their simplest forms. These include the observation that biochemical and morphological degenerative changes observed in fibroblasts aged in vitro do not resemble alterations observed in cells obtained from aged donors, and the fact that tissues transplanted serially through different hosts do not decline in vigor in the manner predicted by the somatic mutation theory. Although biochemical and mutational alterations appear to accumulate in fibroblasts aged in vitro (in support of the error catastrophe model), there are substantial problems with the interpretation of such experiments, and some observations (such as the lack of increase in translational error in hemoglboin synthesis as a function of age) seem to argue directly against the error catastrophe theory. Some alternative theoretical and experimental possibilities are discussed, including the concept of programmed aging as the cause of senescence.

Mutagenesis studies on cultured mammalian cells. The sensitivity of the asparagine-requiring phenotype to several chemical agents ☆

The effect of several chemical agents on the mutation frequency from asparagine dependence to asparagine independence has been studied in Jensen sarcoma cells. It was found that ethylmethanesulfonate brought about a dramatic exponential increase, while nitrosoguanidine was not lighly effective as a mutagen, causing only a modest increase in mutation frequency, and quinacrine HCl was ineffective. The results presented here are compared with those obtained in other systems and with our previous work on the effects of UV on mutation induction in the asparagine system. They suggest that the basis of the asparagine requirement of mammalian cell lines resides in a specific genetic alteration in nuclear DNA which is corrected by the mutagenic action of the agents tested here.

Ultraviolet-induced mutations to asparagine independence in Jensen sarcoma cells in vitro

Abstract UV-irradiation induces an exponential increase in the frequency of mutation from asparagine requirement to asparagine non-requirement in Jensen sarcoma cells grown in vitro . The corrected mutation frequency increases from the spontaneous rate of 5.1·10 −6 per cell to 1248·10 −6 per cell with a dose of 180 erg/mm 2 of 254 nm UV A substantial increase was oberved even without correction for survivors, and no significant difference was observed in the UV sensitivity of asparagine-requiring and non-requiring Jensen clones. When Jensen cells were plated at low densities in a feeder layer of LMTK-cells inactivated by HAT medium, an increase in the cloning ability of the former was observed as compared to appropriate controls without the feeder layer, but the increase was constant over all doses of UV tested. Revertants are stable and possess measurable asparagine synthetase. It is concluded that UV is an extremely effective mutagen in this system.

On the relationship between spontaneous mutation rates in vivo and in vitro.

Recent estimates of spontaneous mutation rates in man, in which previous sources of bias are corrected, indicate that the average is about 3 x 10(-7) per locus per generation, a much lower figure than is generally accepted. Assuming 100 to 1000 cell divisions between each gametic union, this information predicts that cellular mutation rats should be in the order of 10(-9) per locus per generation. Since none of the mutation rates measured in cultured cells are this low (average for seven characters equals 7 x 10(-7)), the size of mutation rates in cultured cells cannot be used to substantiate the claim of epigenetic inheritance. Furthermore, this information suggests that in multicellular organisms the germinal tissue is sequestered from mutagenic insult or subjected to selection against mutational damage so as to keep the genetic load of a species at a tolerable level. Alternatively, cell culture environments may present an extremely abnormal situation to somatic cells, thus elevating the mutation rate.

Puromycin resistance in Chinese hamster cells: Genetic and biochemical studies of partially resistant, unstable clones

Resistance to 10 microgram/ml of puromycin has been analyzed in V79 Chinese hamster cells. Clones that were isolated in 10 microgram/ml of puromycin and subsequently cultivated in its absence consistently lost their resistance. One clone was analyzed in detail by recloning in the presence and absence of puromycin, and it was found that non-puromycin cultivated subclones also lost their resistance and regained inhibition profiles similar to the V79 parent. Reconstruction experiments between sensitive and resistant cells demonstrated that the yield of mutants was not affected by metabolic cooperation. The mutation rate was calculated to be 1 x 10(-7) per cell generation, and was the same within the limits of statistical error in a colchicine-produced polyploid derivative of the V79 line. Although a number of resistant clones were found to have polyploid karyotypes, the polyploid V79 lines was not more resistant to puromycin, nor did it possess a higher frequency of puromycin resistant cells. Studies employing radiolabeled puromycin established that resistance was due to a lowered uptake of puromycin and that an inverse relationship existed between resistance level and uptake rate.

Aldehyde dehydrogenase isozymes in rat hepatoma-mouse fibroblast hybrids.

A study of aldehyde dehydrogenase in rat hepatoma cells and rat hepatoma-mouse fibroblast hybrids revealed that the hepatoma cells had activity comparable to that found in whole rat liver and that the enzyme activity was suppressed in early hybrids and reappeared following chromosome loss. Starch gel electrophoresis and heat inactivation studies showed that a new form of enzyme was produced in the hybrids, possibly a heteropolymorphic combination between the HTC enzyme and a previously repressed mouse form. Staining methods for starch gel electrophoresis and histochemical detection of aldehyde dehydrogenase are described.

The effect of nerve growth factor on dispersed neuronal-Hela heterokaryons

Abstract Dorsal root ganglia from 7-day chick embryos were minced and slowly trypsinized to produce a suspension of cells. This suspension was centrifuged in an albumin density gradient and the fraction enriched with neuronal perikarya collected. This fraction was combined with an equivalent number of HeLa cells. The two cell types are easily identifiable with interference microscopy. The two cell types were fused by the addition of β-propiolactone-inactivated Sendai virus. The resultant heterokaryons were cultured in modified hanging drop preparations in the presence of nerve growth factor and supplemented medium. Nerve growth factor stimulated the growth of long processes from the neuronal-HeLa heterokaryons when added to both long- and short-term cultures thereby demonstrating retention by the heterokaryon of a differentiated property of the neuron. We conclude that differentiated properties can be retained in somatic cell hybrids.