Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. I. Properties of aluminium X-rays and preliminary experiments with Chinese hamster cells.

Irradiation with ultrasoft X-rays produces electron tracks of short defined lengths in the irradiated material. This property is of particular interest in distinguishing between different models of radiation action on living organisms. The production, absorption and dosimetry of aluminium K characteristic X-rays of energy 1.5 keV are described. Quantitative experiments on mammalian cells with these X-rays are possible, and they were found to be considerably more effective than gamma-rays in inactivating Chinese hamster V79 cells in vitro.     

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. III. Implication for theory of dual radiation action.

Microdosimetric distributions for aluminium K characteristic ultrasoft X-rays and 4He ion tract intersections are calculated and used to analyse recent biological results obtained with these radiations. Results on inactivation and mutation-induction to thioguanine resistance of both V79 Chinese hamster cells and HF19 human diploid fibroblasts in vitro are analysed in terms of the Kellerer-Rossi "theory of dual radiation action". The small quantum energy of the aluminium X-ray photons and the very short length of the secondary electrons which they produce highlight the inadequacy of the model. It is shown that the model predicts r.b.e. values in conflict with those observed unless an additional variable is introduced, but that the introduction of such a variable creates mathematical inconsistencies. The experimental evidence is contrary to the conventional usage and basis of the model.     

Effectiveness of 1.5 keV aluminium K and 0.3 keV carbon K characteristic X-rays at inducing DNA double-strand breaks in yeast cells

Induction of DNA double-strand breaks in diploid wild-type yeast cells, and inactivation of diploid mutant cells (rad54-3) unable to repair DNA double-strand breaks, were studied with aluminium K (1.5 keV) and carbon K (0.278 keV) characteristic X-rays. The induction of DNA double-strand breaks was found to increase linearly with absorbed dose for both characteristic X-rays. Carbon K X-rays were more effective than aluminium K X-rays. Relative to 60Co gamma-rays the r.b.e.-values for the induction of DNA double-strand breaks were found to be 3.8 and 2.2 for carbon K and aluminium K X-rays respectively. The survival curves of the rad54-3 mutant cells were exponential for both ultrasoft X-rays. For inactivation of rad54-3 mutant cells, the r.b.e.-values relative to 60Co gamma-rays were 2.6 and 2.4 for carbon K and aluminium K X-rays, respectively. The DNA double-strand break data obtained with aluminium K and carbon K X-rays are in agreement with the data obtained for gene mutation, chromosome aberrations and inactivation of mammalian cells, suggesting that DNA double-strand breaks are the possible molecular lesions leading to these effects.     

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. II. Dose-responses of Chinese hamster and human diploid cells to aluminium X-rays and radiations of different LET.

The induction of inactivation and mutation to thioguanine-resistance of two types of cultured mammalian cells, V79 Chinese hamster and HF19 human diploid, was studied after irradiation with aluminium K characteristic ultrasoft X-rays, helium ion track intersections of different LET, 42 MeV d-Be neutrons, and hard X- or gamma-rays. The form of the dose-response curves was different for the two cell-types, and there was an overall difference in radiosensitivity, the human cells being the more sensitive to all radiations. However, for both inactivation and mutation-induction, the relative responses of both cell-types to these radiations was similar. Aluminium X-rays were considerably more effective than hard X- or gamma-rays and were at least as effective as helium ions of 20-28 keV micron-1, although aluminium X-rays produce tracks of very limited range (less than about 0.07 micron). Single track effects by aluminium X-rays cannot, therefore, extend beyond about 0.07 micron, and the subcellular sites involved in inactivation and mutation cannot be greater than this dimension or else the effectiveness of aluminium X-rays would be similar to that of low-LET radiations. This observation is in contradiction to models of radiation action which require relatively large sensitive sites; for example the 'theory of dual radiation action' requires a site diameter of about 0.4 micron to explain the shape of the dose-response curves for V79 hamster cells.     

The induction of chromosome exchange aberrations by carbon ultrasoft X-rays in V79 hamster cells

V79 hamster cells in plateau (extended G1) phase were irradiated with either 250 kV ('hard') X-rays or carbon K characteristic ultrasoft X-rays under conditions minimizing cell overlap. These cells were killed most effectively by the carbon X-rays, by a factor of about 3 relative to hard X-rays, in agreement with our previous findings with cells in exponential growth. Chromosome-type aberrations were measured at 3 fixation times within the first division cycle after irradiation, and an approximately uniform sensitivity to aberration induction was found for both radiations. The combined aberration data show that carbon X-rays are 2 or more times as effective as hard X-rays, depending on dose and/or data fit. Exchange aberrations require recombination between two separate chromosomes, but they are induced efficiently by carbon X-rays with a substantial linear component to the dose-response despite the very short electron tracks (approximately less than 7 nm) that they produce in the cell. This implies either that the participating DNA helices must be lying extremely close together at the time of radiation damage, so that one track can effectively damage both helices, or that only one radiation-damaged chromosome is needed to promote an exchange event.     

Estimation of mutation rates in cultured mammalian cells

The factors that affect reliable estimations of mutation rates (μ) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (N_t) and the number of parallel cultures (C) on the variation in the rate estimates ($\text{μ}$) inferred from the P₀ method. The analysis can be made after the derivation of the variance of $\text{μ}$, which is a measure of variation of $\text{μ}$ for a given combination of N_t and C in a number of repeat experiments. The variance of $\text{μ}$ is inversely proportional to C and to the square of N_t. N_t determines the probability of occurrence of mutation in a cell culture. By influencing the size of P₀, N_t also determines whether a rate estimate is obtainable from the experiment. Since P_o is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of $\text{μ}$. The rate estimated from $\text{P}\text{?}{}_0$ is biased, but the bias is in general 2 orders of magnitude smaller than $\text{μ}$. By the selection of an appropriate combination of N_t and C for the experiment, this bias can be reduced even further.Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.     

Cell-cycle dependency of BUdR-induced mutation for six genetic markers in cultured mammalian cells.

Synchronized mouse L5178Y cells were treated with BUdR during each one of four sequential periods of the cell cycle (M-G1, early S, middle S and late S-G2). Among the 6 markers examined, asparagine independence (Asn+), 6-thioguanine resistance (TGr) and excess thymidine resistance (TdRr) showed maximal induction of mutation rates in the early S period, methotrexate resistance (MTXr) gave maximal induction during the middle S period, and two other markers [arabinosylcytosine resistance (Ara-Cr) and ouabain resistance (Ouar)] showed little mutation induction in any period under the experimental conditions. These results suggest that (i) genes responsible for Asn+, TGr and TdRr activity may be replicated in the early S period and the gene for MTXr activity replicated in the middle S period, and (ii) the mechanisms of mutation induction for the Ouar and Ara-Cr markers may be essentially different from those for the Asn+, TGr, TdRr and MTXr markers.     

A deterministic approach for the estimation of mutation rates in cultured mammalian cells

Unequal growth rates between mutant and wild-type cells in a large population constitute a problem for the estimation of mutation rate. Over a period of cell growth, a selective advantage of one cell type over the other might lead to considerable error in the estimation of mutation rate if equal growth rates are assumed. In this study, we propose a formula and apply it to the estimation of spontaneous mutation rate in a growing population of Chinese hamster V79 cells in which ouabain-resistant mutant cells exhibit a slower growth rate than the wild-type cells. The formula is a generalization of that previously presented by Armitage (1953), and this is the first attempt to apply the deterministic approach for mutation rate estimation to cultured mammalian cells. The value of the estimated rate is compared with that derived from a parallel experiment using the fluctuation test of Luria and Delbrück (1943). The limitations and advantages of taking the deterministic approach to mutation rate estimation in mammalian cell systems are discussed.     

Induction of fatty acid synthesis in cultured mammalian cells: effects of cycloheximide and X-rays

It was found that L cells deprived of exogenous fatty acids responded adaptively by increased synthesis of fatty acids. This adaptive increase was found to be sensitive to cycloheximide presented at the time of removal of exogenous lipids, but was not sensitive to cycloheximide added six hours after removal of lipids. It was concluded that the adaptive increase in fatty acid synthesis represents a case of enzyme induction. The effect of x-rays on this process was studied. It was found that induction was inhibited to a greater extent than total protein synthesis. Factors of potential importance to such an effect are discussed.     

Anticlastogenicity in cultured mammalian cells.

Basic and applied research on anticlastogenicity has not only revealed valuable evidence on the mechanisms governing the induction of chromosomal aberrations by environmental mutagens, but also contributed effective ideas on a practical employment of this knowledge for the protection of individuals at risk. Considering the basic role played by chromosomal anomalies in oncogenesis, additional weight must be attributed to studies on anticlastogenicity. The employment of human cells in this kind of study dates back to 1969/70, while classical mammalian cell systems were used only later on. Various modes of application of both clastogens and anticlastogens (AC) were examined, but simultaneous addition to the cultures of both reagents was the most favored way. A wide spectrum of cytogenetic endpoints can be studied, but differences can be demonstrated with regard to efficacy of inhibitors on different types of cytogenetic changes, e.g., open breaks vs. rearrangements, but also vs. SCEs. Depending on their mode of influence on this spectrum, ACs can be arranged in various categories which are of practical importance, for instance, with regard to their oncogenic potential. A wide variety of factors was shown to influence AC action, e.g., time and mode of application of the test substances, physiologic and metabolic features of the cell types studied, type and mechanism of the clastogen used, etc. The addition of S9 mix can drastically change the patterns of efficacy of the ACs. The combined application of two or more ACs, as far as investigated, apparently neither potentiates nor even merely adds their effects.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. II. Chinese hamster V79 cells.

Inactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV micrometers-1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0.20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90-200 keV micrometer-1. However, the maximum mutagenic effectiveness relative to gamma-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.     

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. III. Human diploid fibroblasts.

The induction of inactivation and mutation to thioguanine-resistance in cultured human diploid fibroblasts was studied after exposure to ionising radiations with LET's in the range 20--470 keV micrometer-1. Unique r.b.e. values were obtained for inactivation and mutation induction with nine different qualities of radiation. The plot of r.b.e. verus LET gave humped curves for both endpoints; r.b.e. maxima were in the LET range 90--200 keV micrometer-1 but the maximum r.b.e. value for mutation induction was almost twice that for inactivation. The accuracy of estimates of mutation induction are discussed with regard to possible selective effects against mutants during post-irradiation growth.     

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. I. Irradiation facilities and methods.

This paper, which is the first of four covering the inactivation of clonogenic capacity and induction of mutation in cultured mammalian cells, deals briefly with the general aims of the work and describes the irradiation techniques used. Human diploid fibroblasts and V79 Chinese hamster cells were irradiated as monolayers with ions of helium, boron or nitrogen at LET's in the range 20 to 479 keV micrometer-1 in H2O. The physical aspects of the irradiation including measurement of ion energies, dosimetry and uniformity of dose and also the methods of handling large numbers of samples are described in detail. Subsequent papers will present the biological methods and results and a biophysical analysis of the data.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Evidence for inactivation of DNA repair in frozen and thawed mammalian cells.

A variety of cell strains and lines were frozen and thawed by conventional techniques for cell storage. Following thawing, extracts of cells were prepared and incubated with UV-irradiated E. coli DNA. Thymine dimer excision activity present in extracts of unfrozen cells was lost in extracts of recently thawed cells. The ability to exercise dimers was restored after about 40 h post-thawing, but the recovery was inhibited if cells were cultured in the presence of puromycin. Correlating with the loss of dimer excising activity there was a reduced cell viability as measured by trypan blue dye exclusion.     

A method for enucleating cultured mammalian cells

A method is described for enucleating cells which normally could not be enucleated due to their poor adhesion to the growth surface. The technique consists of linking ConA to the surface and then applying the cells. This results in cell adhesion firm enough to withstand the centrifugal forces necessary to enucleate. The method has been applied to fibroblastic, epithelioid and lymphoid cell lines.     

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.     

Ribonuclease of cultured mammalian cells

KB cell ribonuclease has been purified 260-fold and the fundamental properties have been studied. Though the enzyme is concentrated in the lysosomal fraction, appreciable quantities are present in the cell sap and nuclear fractions. Comparison of the optimal temperature and pH for activity, and the heat stability of enzyme from these three fractions suggests that only one species of this enzyme exists in these cells. The enzyme behaves as an endonuclease, cleaving synthetic pyrimidine polynucleotides to smaller oligonucleotides with cyclic 2′:3′ end-groups. The final product is pyrimidine nucleoside 3′ monophosphate. Polyadenylic acid is not hydrolyzed. Of the properties examined in this study only two differences were noted between KB cell and pancreatic ribonuclease. KB cell enzyme acts optimally at pH 6 as opposed to an optimum at pH 7 to 8 for pancreatic enzyme. In addition ribonuclease from KB cells is definitely less stable to heating at 100°C than is the enzyme isolated from pancreas.