The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.     

The relationship between chromosomal aberrations, survival and DNA repair in tumour cell lines of differential sensitivity to X-rays and sulphur mustard

A pair of cultured rat lymphosarcoma cell lines (Yoshida) with a pronounced differential sensitivity to killing with sulphur mustard (SM), but with the same sensitivity to X-rays, was examined for chromosome damage and DNA repair replication after treatment with these agents. A pair of mouse lymphoma cell lines (L5178Y) with a differential sensitivity to X-rays was similarly investigated.SM-resistant Yoshida cells suffered much less chromosome damage than sensitive cells in spite of equal alkylation of DNA, RNA and protein in sensitive and resistant cells. The pair of Yoshida cell lines sustained the same amount of chromosome damage after X-irradiation. Much less chromosome damage was observed in the radiation-resistant lymphoma cell line than in the sensitive line after X-irradiation.No differences was found between the pairs of cell lines in their capacities for repair replication after SM or X-ray treatment.Thus, the drug and radiation resistance is accompanied by, and perhaps mediated through, a reduced amount of induced chromosome damage but is not quantitatively related to the capacity for DNA repair replication.Apart from small differences in modal chromosome numbers there are no obvious karyotype differences between the sulphur mustard-sensitive and -resistant Yoshida cells or between the radiation-sensitive and -resistant lymphoma cells.     

Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents

Repair replication has been measured by CsCl density gradient centrifugation in cell lines showing differential sensitivity to mono- and bifunctional alkylating agents. A correlation between cellular sensitivity as measured by the D₀ value and amount of repair replication was demonstrated after exposure of Yoshida cells to nitrogen mustard (HN2) and methylene dimethanesulphonate (MDMS). No differences in the amount of repair replication after methyl methanesulphonate (MMS) were observed in two L5178Y cell lines which differed in sensitivity by virtue of the shoulder size only. The Yoshida cell lines showed no difference in sensitivity to MMS and no difference in amount of repair replication. Incorporation of tritiated thymidine 9[³H]TdR) after drug treament was also measured by autoradiography. The qualitative differences observed between the two cell lines were similar to those obtained in density gradient experiments. The temporal pattern of [³H]TdR uptake indicated that the reduced repair replication observed in the sensitive line after HN2 and MDMS is not due to slower synthesis. The kinetics of [^3H]TdR incorporation differed for all three mutagens suggesting that different enzymes may be involved in each case.     

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.     

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 µCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [³H]deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [³H]deoxyadenosine selection, lymphoma cell clones resistant to 15 µCi/ml [³H]deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [³H]Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

Isolation and characterization of an adherent, 8-azaguanine resistant variant of the Burkitt lymphoma cell line, Raji

A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition, susceptibility to Epstein-Barr virus (EBV) infection and inducibility of latent EBV. In order to facilitate fusion experiments with Raji-A, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8-azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 50 μg/ml of 8-azaguanine. They had only 10–15% as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of 1–3 × 10⁻⁶ in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.     

Studies on 1-beta-D-arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts. II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK^?) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK^? cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK^? hamster line and TK^? lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.     

Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation

The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.     

Studies on 1-β- -arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts : II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK⁻) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK⁻ cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK⁻ hamster line and TK⁻ lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Differential spermatogonial stem-cell survival and mutation frequency

One group of adult C3H×101 hybrid male mice was given 3 injections of 12.5 μCi of [³H]thymidine at 9-h intervals and irradiated 24 h after the last injection with X-ray doses of 100, 300, 500, 600, 1000 R or the first fraction of a split 1000-R dose given as two 500-R exposures 24 h apart. Mice were killed 207 and 414 h after irradiation. A second group of mice was given a single injection of 12.5 μCi of [³H]thymidine 1 h before irradiation with single exposures of 300, 500, 600, 1000 R, or the first fraction of a 1000-R exposure given as two 500-R fractions 24 h apart. Mice were killed 120 and 207 h after irradiation. In both experiments, parallel groups of mice were given X-ray only as a control for the effect of [³H]thymidine. Two sets of slides were prepared for each mouse receiving [³H]thymidine: one set was not autoradiographed and was used for scoring cell survival; the second set was coated with emulsion and used for scoring percentage of labeled cells. The dose-response curves for survival at 120 and 207 h were curvilinear, with no evidence of discontinuity over the 100–1000-R range. After multiple injections of [³H]thymidine and irradiation 24 h later, percentage of labeled cells at 207 h was comparable for controls, 100, 300, and 600 R; significantly lower than controls for 1000 R; and significantly above controls after 500 + 500 R. Thus the surviving stem-cell population was qualitatively the same for that portion of the dose-response curve giving a linear increase in mutation rate but was different for both 1000-R and 500 + 500-R exposures, and the single and fractionated 1000-R exposures differed from each other. This parallelism between survival of labeled cells and mutation frequency in spermatogonial stem cells suggests that a stage in the cell cycle 24–42 h after DNA synthesis is resistant to cell killing but sensitive to mutation induction. The mutation rate after a single 1000-R exposure is low because labeled, mutation-sensitive cells have been selectively killed. Mutation frequency after the 500 + 500-R dose is increased because of synchronization induced by the first dose combined with selective killing of unlabeled cells by the second fraction. Irradiation 1 h after labeling with [³H]-thymidine demonstrated that the S phase of the spermatogonial stem-cell cycle is sensitive to radiation-induced cell killing.     

Reassociation of human lymphoblastoid cell DNA repair replicated following methyl methanesulfonate treatment

The reassociation rates of repair replicated DNA of two human lymphoblastoid cell lines, the WIL₂-A3 ‘normal’ line and the RAJI line of Burkitt's lymphoma, were examined using the DNA/DNA ‘C₀t’ hybridization technique. The cells were treated with methyl methanesulfonate (MMS), an alkylating agent and mutagen, to induce the repair.The incorporated repair replication radioactivity in highly repetitive sequences of WIL₂-A3 cell DNA reassociates as expected for a randomly distributed incorporation. The reassociation of repair radioactivity in sequences of fewer numbers of copies, however, is less than expected for a random distribution. It is less than that occurring for semiconservatively synthesized DNA of WIL₂-A3 cells co-incubated with the repair labeled DNA as an internal control.The observed difference could be due to an over-representation of repair replication radioactivity in DNA sequences with fewer copies. It is unlikely to be due to residual alkali labile damage resulting from MMS treatment, since a similar difference was not observed when semiconservatively labeled DNA from cells which had been treated with MMS for the same time and at the same concentration as in the repair experiments was substituted for repair replicated DNA in the reassociation reactions. Other possible causes of the apparent difference in the reassociation rates observed are discussed.     

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.     

A study of the effect of caffeine upon excision repair of damaged DNA.

Cultured mammalian cells incur damage to their DNA when exposed to ultraviolet light or adduct-producing mutagens such as 4-nitroquinoline-1-oxide (4NQO). At least two processes are important in repair of such damage: post-replication repair and excision repair. Many researchers have reported that caffeine inhibits the former process, which occurs in connection with semiconservative DNA replication, especially in rodent cell lines such as mouse lymphoma or Chinese hamster. Excision repair is not generally considered caffeine-sensitive, although the data are somewhat conflicting because some studies had used rodent cells, which show little or no excision repair, or human cells in which alternate repair processes may have been operating.Human peripherhal blood lymphocytes from healthy donors were treated with UV light or 4NQO in order to produce pyrimidine dimers or adducts. Caffeine at concentrations of 0.75–3.0 mM was included in some cultures. The cells treated with caffeine were incubated for 90 min prior to mutagen treatment and for the entire period thereafter until cell harvests. [³H]Thymidine was added and the uptake quantitated as a measure of DNA repair. DNA replication was inhibited by hydroxyurea, so that only excision repair was measured by this method. Separate plates of cells not exposed to mutagens exhibited negligible or low thymidine uptakes.Following harvest, the cells were lysed and the DNA extracted. The DNA released was measured spectrophotometrically and then placed into liquid-scintillation counter (LSC) vials for measurement of incorporated radioactivity. Resulting cpm/μ DNA were compared for cells with and without caffeine. Lymphocytes from patients with systemic lupus erythematosus (SLE), who previously had demonstrated reduced levels of excision repair under these conditions, were also tested with caffeine. Caffeine did not inhibit repair by normal lymphocytes and the reduced repair seen in the SLE patients was not further reduced in its presence.In a series of pulse-chase experiments, some cells were treated with 4NQO and allowed to incubate with [³H]thymidine for 3 h and were harvested at the end of this period, while others were given a 13-h chase i n cold thymidine before harvest. The cpm/μg DNA for both groups were virtually identical, both in the presence and absence of 2.0 mM caffeine.     

Amphibian cells in culture. II. Isolation of drug-resistant variants and an asparagine-independent variant.

With L-15 as the base medium, drug-resistant variants were isolated from two amphibian tissue culture strains: the Xenopus laevis A8 diploid cell line and the ICR 2A cell line of Rana pipiens. Four different classes of variants were obtained: (1) A8 cells resistant to chloramphenicol, an inhibitor of mitochondrial protein synthesis; (2) A8 cells resistant to ouabain, an inhibitor of the Na+/K+-activated ATPase of the plasma membrane;(3) ICR 2A cells resistant to low (20 microgram/ml) and high (300 microgram/ml) levels of bromodeoxyuridine (BUdR), a thymidine analog which interferes with the pyrimidine salvage pathway; and (4) ICR 2A cells resistant to 2,6-diaminopurine (DAP), an adenine analog which interferes with the purine salvage pathway. Unlike the other variants, isolation of BUdR resistant cells is a 2-step process. Resistance to low levels of BUdR is phenotypically expressed by a reduction in thymidine transport activities while resistance to high levels of this compound is evidenced by greatly reduced levels of thymidine kinase activity. DAP-resistant cells, which are characterized by reduced levels of adenine phosphoribosyl transferase (APRT) activity, do not die in AAT (adenine, aminopterin, thymidine) selection medium. This suggests that these cells utilize adenine efficiently. With MEM as the base medium, an asparagine independent clone was isolated from the ICR 2A cell line. When compared with the wild type, this variant exhibited a slightly reduced growth rate in the presence or absence of asparagine.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

Induced radioresistance: An aspect of induced repair

Summary Irradiation of Escherichia coli cells with UV or X-rays followed by incubation under conditions in which protein synthesis can occur results in a population of cells that is resistant to X-rays; however, this resistance develops only if the cells are recA ⁺ and lexA ⁺, a fact that associates the phenomenon with induced (S.O.S.) repair. By observing separately the component of a culture that is resistant and the component that retains its normal growth, the fraction of induced and uninduced cells for a dose of UV or X-rays can be estimated. Such estimates show that the dose-response for UV induction of resistant cells agrees with that of the recA gene product. Thus induced radioresistance is considered to be due to the changes in the cell occasioned by the derepression of recA and lexA. These changes are expected to be involved with the synapsis of homologous genomes that is necessary for the use of a second genome to repair damage occurring in both strands of a duplex at the same base, as exemplified by a double-strand break or an interstrand crosslink. This consideration is additionally supported by the increased resistance of cells grown to contain multiple genomes in the same envelope, an increased resistance not found in recA ^- or lexA ^- cells. The condition of a completed chromosome is also resistant, again not in recA ^- or lexA ^- cells. We suggest that cell killing by X-rays is due to the double-strand breaks which are not repaired by molecular synapsis before the arrival of the replication polymerase at the break.     

The dose-response relationships for myeloid leukemia and malignant lymphoma in BC3F1 mice

The present analysis of data on the induction of lymphoma and myeloid leukemia in BC3F1 mice has indicated some new and interesting aspects regarding the shapes of the dose-effect curves. The incidence data can be interpreted by radiobiological models of the induction process coupled with cell inactivation. In particular, for malignant lymphoma the dose-response curve after X rays can be described assuming a quadratic model corrected for cell inactivation, while the incidence data after fission neutrons are best fitted by a linear model which also allows for cell inactivation. Myeloid leukemia has also been induced in BC3F1 mice. The bell-shaped dose-response curves observed after irradiation with either X rays or neutrons are explained by assuming simultaneous initial transforming events and cell inactivation with the data for cell inactivation at higher doses being in agreement with data reported for other strains of mice. A value for relative biological effectiveness of 4 is obtained at the lowest neutron dose used. The value of the inactivation parameters can be compared with those of the cell inactivation probability per unit dose for the bone marrow hematopoietic stem cells, which are believed to be the target cells for these tumors.     

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.