Arthur D. Bloom

Induced Chromosomal Aberrations in Man

It has become increasingly clear in recent years that a multiplicity of chemical and physical agents to which man is exposed are capable of damaging his chromosomes. The effects of many such agents on human somatic cell chromosomes have now been well documented, albeit not without occasional controversy over the interpretation and implications of the data. While the biological and clinical significance of chromosomal aberrations are not as yet well understood, recent epidemiological evidence, from mutagen-exposed human populations, and recent experiments on viral-induced transformation of chromosomally abnormal cells suggest an association between chromosome aberrations and oncogenesis.

Lactic Dehydrogenase and Metabolism of Human Leukocytes in vitro

During transformation and division of lymphocytes in culture, the lactic dehydrogenase isozymes migrate increasingly toward the cathode. With extension of the time in culture, the mitotic index declines, and the isozyme pattern reverts to dominance of those bands that move toward the anode, despite the cellular tendency to anaerobic metabolism. These findings suggest that synthesis of the more slowly migrating lactic dehydrogenase isozymes in this system is related to mitotic activity, and not to the aerobic or anaerobic conditions of cell culture.

Immunoglobulin Production by Human Lymphocytoid Lines and Clones: Absence of Genic Exclusion

Immunoglobulin production was studied in established lines of normal human lymphocytes. Three lines which produced both immunoglobulin G and immunoglobulin M were cloned. Among the 25 immunoglobulin-producing clones, 23 produced both classes of immunoglobulins. These findings suggest that the phenomenon of genic exclusion does not hold for immunoglobulin production in lymphocytoid cells in culture.

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7±8.7 nmoles per hour per milligram of protein and an apparent Km for citrulline of 2 × 10−4m, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7±3.8 nmoles/hr/mg and an apparent Km for citrulline of 2 × 10−2m. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.

Citrulline Metabolism in Normal and Citrullinemic Human Lymphocyte Lines

Citrullinemia is one of the five aminoacidurias associated with the Krebs-Henseleit urea cycle. A long-term lymphocyte line (UM-21) derived from a patient with this disease and nine of ten clones of this line were found to have no activity for the enzyme argininosuccinate synthetase (AS), as demonstrated by their inability to grow in medium in which citrulline had been substituted for arginine, by their inability to incorporate arginine-C14 derived from citrulline-C14 into cellular protein, and by direct enzyme assay. One clone had normal or nearly normal argininosuccinate synthetase activity, as demonstrated by the same criteria. Nutritional “variants” able to grow logarithmically in medium containing citrulline were isolated from UM-21 and three clones. The apparent Kms of AS for citrulline in UM-21, the ten clones, the variant lines, and a normal line were measured and fell into three groups: AS in UM-21 and nine clones had no measurable apparent Km for citrulline; AS in the variant cells had apparent Kms for citrulline of approximately 20 mm; and AS in the normal cell line and one clone had apparent Kms for citrulline of 0.2 mm. The data suggest that the defect in the citrullinemic cell lines is due to a mutation in the structural gene coding for argininosuccinate synthetase.

Induced chromosomal aberrations: Biological and clinical significance

A multiplicity of chemicals, viruses, and ionizing radiations are now known to damagethe genetic material of human cells. Chromosomal breaks and rearrangements are relatively gross measures of such damage. However, there is evidence that increases in chromosomal aberrations may be seen in human populations which are at high risk in terms of neoplastic disease. Further, studies of chromosomally abnormal cell populations in vitro have revealed an increased tendency to malignant transformation after exposure to oncogenic DNA viruses. The clinical and biological significance of induced aberrations in man is reviewed in the light of these findings.

Heat Lability of NADPH at Physiologic pH

Recently, the procedure of induced fluorescence (Lowry et al., 1957) was adapted for the microassay of glucose 6-phosphate dehydrogenase (G6PD) in preimplantation mammalian embryos (Brinster, 1970). In this assay, samples were incubated in the presence of NADP, glucose 6-phosphate, MgC1/, EDTA, and crystalline bovine serum albumin in tris-HC1 buffer (pH 7.5) for periods of 30 and 60 min, and the enzymatic reaction was terminated by immersing the sample tubes in boiling water for 2 min. Succeeding steps involved destruction of the excess NADP with dilute NaOH (Lowry et al., 1957) and the production of a fluorescent product by a mixture of strong base and hydrogen peroxide. In the course of our investigation of G6PD activities in preimplanted rabbit embryos, inconsistent results obtained by using the above method led us to examine the individual steps of the procedure. It is well known that NADPH is stable at alkaline pH (Lowry et al., 1961). We report here our findings on the temperature lability of NADPH at physiological pH. Solutions of NADPH (Sigma Chemical Company) were prepared in a concentration range suitable for direct fluorometric analysis (1 x 10-6 to 9 x 10 .6 N) using 0.05 M tris-HC1 buffer at pH 7.5. Aliquots of these solutions were placed in a water bath at 22, 60, or 100 C, and after 0.5, 1, 2, 4, 8, and 16 min the tubes were transferred to an ice bath. After warming to room temperature, the native fluorescence of NADPH was recorded in an AmincoBowman spectrophotofluorometer (SPF). In separate experiments, the complete enzyme assay was done using

MUTAGENESIS IN HUMAN LYMPHOID LINES

We are attempting to develop the human lymphocyte culture system for study of rates and mechanisms of chemically-induced mutation. Three loci are being examined: the hpt locus which specifies HGPRT synthesis; the as locus which specifies argininosuccinate synthetase (AS) synthesis; and the o locus which determines ouabain responsiveness. Mutation from hpt+→hpt−, using 6-thioguanine (6TG) selection, is being studied, as is mutation from as−→as+, using high citrulline-low arginine medium, and Os→Or, using 106M ouabain. The tester line UM-21-5, of citrullinemic (as−/as−) origin, has, via selection, produced a line triply mutant (hpt−, as−, and Or), as determined by growth in selective media and by HGPRT and AS specific activities. The Wi-L2 tester line, derived originally from a spherocytosis patient, has produced, by selection, an hpt− and as− derivative. Spontaneous mutation rates in Wi-L2 from 6TtS→6TGr(hpt+→hgt−), via fluctuation analyses, ranged from 0.1-0.3 × 10−7cell/generation. Using sister chromatid exchange (SCE) frequency as an indirect measure of mutagenicity, incubation of Wi-L2 in ethyl methanesulfonate (EMS), 50 μg/ml for 24 hours, resulted in a 3-fold increase in SCEs (mean in controls of 7.7 SCEs/taetaphase vs. 21.9 in EMS-exposed cells). The mutation rates at these 3 loci after EMS mutagenesis are now being determined, as a model for demonstrating the feasibility of monitoring for chemical mutagens using these lymphoid lines.

HYBRIDIZATION OF ESTABLISHED IMMUNOGLOBULIN-PRODUCING CELLS

To study the regulation of immunoglobulin (Ig) production in human lymphocytes and to link the genes for heavy and light chain synthesis to specific human chromosomes, we have developed methods for hybridization of the cells of established lymphocyte lines. Our first fusion involved an interspecific human lymphocyte (HGPRT-, kappa chain producing) × hamster fibroblast (TK-) cross; the second, an intraspecific human lymphocyte × lymphocyte cross, with both lines producers of gamma, mu, and kappa chains. In both instances, the lymphocytes were pre-treated with 0.01% trypsin prior to addition of Sendai virus. In the interspecific cross, kappa chain production was lost; in the intraspecific cross, in which the chromosomes of both parental cells were initially retained, gamma, mu, and kappa chain production were also retained, as determined by immunofluorescence. The lines resulting from the lymphocyte × lymphocyte hybridization were, however, unstable, and reverted to diploidy by 8 weeks post-fusion. We are currently attempting to establish and maintain a chromosomally stable intraspecific hybrid obtained from fusion of an Ig+ and an Ig− cell line.