Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.     

Studies on three mutagen-sensitive mutants of mouse L5178Y cells. I. Characterization of the hybrids between L5178Y and mutagen-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2'-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen -sensitive mutants. All the TGr lines were sensitive to 5-bromo-2'-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.     

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

Adaptation of mouse leukemic cells (L5178Y) to anisotonic media : II. Cell growth

Mouse lymphoma cells (L5178Y) exposed to hypertonic media for 1 h behave as osmometers, but in hypotonic media, after initial swelling, they shrink back to normal volume and maintain it for long periods of time. The lower limit of osmolarity at which this “volume adaptation” will occur lies between 140 and 185 mosM. The “volume adaptation” is associated with a loss of cellular K⁺ probably due to a transient increase in K⁺ permeability and to loss of associated anions and osmotically obliged water. Partial dissipation of the large gradient of K⁺ between cells and medium by pre-exposure to ouabain or to K⁺-free medium results in a diminished capacity to adapt. After the shrinking phase is completed, a new steady state is established with a reduced cellular K⁺ content, normal Na⁺, normal K⁺-permeability, and a reduced activity of the Na⁺ − K⁺ transport system. When adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K⁺ content, presumably due to the return to normal activity of the Na⁺ − K⁺ transport system.     

Specific G2 phase toxicity of chloramphenicol on mouse leukemic cells (L5178Y)

The effect of chloramphenicol on progression through the cell cycle of L5178Y cells was investigated. Using eosin staining as a viability index, G2 cells were shown to be specifically killed at a concentration of chloramphenicol generally used to study mitochondrial protein synthesis. Pretreating cells with chloramphenicol induced resistance to this G2 lethality.     

Effect of hyperthermia on protein biosynthesis in L5178Y murine leukemic lymphoblasts

The effects of elevated temperatures upon protein biosynthesis were determined in L5178Y murine leukemic lymphoblasts. The rate of protein synthesis was inhibited proportionately to the increase in temperature. Efforts were made to determine the mechanism of heat inactivation of protein synthesis by studying the requirements for recovery of activity after the cells were returned to 37°C. The ability of actinomycin to block the recovery process suggests that elevated temperatures destroy or inactivate a species of RNA required for protein synthesis. Loss of RNA during heating of the cells is apparently at least partially dependent on protein synthesis, since the presence of cycloheximide during heat shock, is capable of ameliorating the effects of short duration heat treatment.     

Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells.

The alkylating agent MMS was toxic to mouse lymphoma L5178Y cells and decreased their growth rate. A dose-dependent induction of thioguanine- and thymidine- but not ouabain-resistant variants was observed. The prolonged period for expression of thioguanine-resistant variants observed with other mutagens was also found in these studies. A comparison of MMS and EMS showed that MMS on a molar basis was approximately 10 times more toxic than EMS. With mutation, however, when evaluated at equal levels of cell killing MMS and EMS induced the same number of thymidine-resistant variants. For thioguanine-resistant variants MMS was approximately 10-fold less efficient than EMS, while for ouabain-resistance MMS, unlike EMBS, produced no variants at all. The ouabain results were further compared with positive results obtained using a modified Luria--Delbrück fluctuation test.     

Effect of chloramphenicol on the growth and viability of exponentially growing mouse leukemic cells (L5178Y)

Two effects of chloramphenicol on mouse leukemic cells (L5178Y) are described. The drug induces a prolongation of the cell cycle (reversible effect). The degree of prolongation is directly proportional to the concentration of the drug. The effect is observed only in the presence of chloramphenicol and cells return to normal cell-growth kinetics when the drug is removed from the culture medium. Chloramphenicol also kills a portion of the cell population immediately (irreversible effect). Data are presented which suggest that chloramphenicol is toxic to cells in the G2 phase of the cell cycle.     

Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells.

The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Cell-cycle dependence of heat-induced interphase death in mouse L5178Y cells.

Cell lysis and eosin staining were observed in L5178Y cells within the first 3 h of post-hyperthermia incubation at 37 degrees C, after which both leveled to a plateau. Lysis and eosin staining were proportional to the severity of heat in asynchronous cells, whereas it was maximum in the most heat-sensitive M phase, intermediate in S, and least in heat-resistant G1 for the same heat treatment. Further, leakage of labeled [3H]thymidine and a decrease in radioactivity retained within heated cells coincided with an increase in eosin staining, indicating that the dye uptake was due to membrane damage. It was presumed that the eosin-stained fraction represented dead cells. The percentage eosin-stained cells reached a plateau, and this level was used to determine survival; when the results were compared with those obtained by the colony formation method, they were identical. By comparing the two survival assay methods we concluded that cell death after hyperthermia in L5178Y cells is mainly by interphase death in all phases of the cell cycle. The reasons for this conclusion are that a reduction in survival could be detected within one generation of L5178Y cells by the eosin staining method, and the survival values obtained by this method were identical to those obtained by the colony formation method.     

Specificity of transport of bleomycin and cobalt-bleomycin in L5178Y cells

The mechanism of transport of [³H]peplomycin (PEP), a new member of bleomycin group antibiotics, was studied in cultured L5178Y mouse leukemic cells. Cobalt ions enhanced the uptake of PEP, but Cu, Zn, Fe(II) and Fe(III) had no effect. The initial rate of uptake of cobalt chelated PEP [PEP(Co)] was several times higher than that of free or Cu-chelated PEP and was temperature independent. A double reciprocal plot of the data demonstrated both saturable (K_m = 4.5 μM, V_max = 1.3 × 10^?18 mole/min/cell) and non-saturable components of the uptake of PEP(Co). The saturable component was inhibited specifically by cobalt chelated bleomycin analogs. PEP-chelates with metals other than cobalt, such as PEP(Cu) were metabolically unstable. These results suggest that bleomycin enters into cells as a metal chelate through a specific transport site.