Thioguanine-resistant Research Articles

Identification of variants of the basophilic leukemia (RBL-2H3) cells that have defective phosphoinositide responses to antigen and stimulants of guanosine 5'-triphosphate-regulatory proteins.

Antigen immunoglobulin E-mediated secretion of histamine from RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and a rise in the concentration of cytosol Ca2+ (calcium signal). Such responses differed among cloned variant lines of the RBL-2H3 cell line from undetectable (1A3 bromodeoxyuridine-resistant (BUDRR), 2B1 BUDRR, and 1B3 BUDRR lines) to about 80% of those in the parent RBL-2H3 cells. In all but one line (1B3 thioguanine-resistant (TgR)), the intensities of the phosphoinositide response and of the calcium signal were correlated with the secretory response. The 1B3 TgR line had no detectable calcium signal (as measured by quin 2 fluorescence or uptake of 45Ca2+) but paradoxically showed modest rates of hydrolysis of inositol phospholipids and of secretion. The responses of the 1B3 TgR line were, however, dependent on the presence of external Ca2+ ions. The induction of secretion with antigen, therefore, was invariably associated with the hydrolysis of inositol phospholipids, but it was not necessarily associated with a change in concentration of cytosol Ca2+. All antigen unresponsive clones could secrete when synergistic signals were induced by exposure to the Ca2+ -ionophore, A23187 and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. These lines, otherwise, had immunoglobulin E receptors and had no obvious defect in their capacity to synthesize the inositol phospholipids or in their phenotypic expression of phospholipase C as measured in cell extracts. One finding of possible relevance to the role of guanosine 5'-triphosphate-regulatory proteins in the activation of phospholipase C was the inability of one antigen-nonresponsive line to respond to NaF (in intact cells) or to guanosine 5'-(3-O-thio)triphosphate (in electrically permeabilized cells).

Detection of the carrier state for an X-linked disorder, the Lesch-Nyhan syndrome, by the use of lymphocyte cloning.

Using a limiting dilution technique, we found that the frequency of thioguanine resistant (TGR) lymphocyte clones was less than 5.0 X 10(-5) in 14 normal individuals, between 9.0 X 10(-3) and 8.9 X 10(-2) in seven heterozygotes for Lesch-Nyhan syndrome, and 0.88 and 0.87 in two hemizygotes. TGR clones from heterozygotes were expanded and had the hemizygote phenotype as evidenced by low hypoxanthine incorporation and severely deficient hypoxanthine-guanine-phosphoribosyl-transferase activity. Enumeration of TGR lymphocyte clones provides a simple technique for detection of heterozygosity for Lesch-Nyhan syndrome. A similar approach using lymphocyte cloning may be suitable for detection of the carrier state for other X-linked disorders.

Measurement of in vivo mutations in human lymphocytes

A means of measuring in vivo mutations in man would be valuable in assessing the importance of mutation in ageing and human disease and for monitoring individuals exposed to environmental mutagens and carcinogens. The hypoxanthine–guanine phosphoribosyl transferase (HGPRT) locus has been used to study mutagenesis in cultured mammalian (and other) cells1; clones are selected by their ability to grow in the presence of the purine analogue, 6-thioguanine (6-TG). Such clones are almost always HGPRT− and are therefore probably the progeny of a mutant cell. This system has been applied to human lymphocytes by using autoradiography to detect single mutant cells2–6. This approach probably does detect mutant cells, particularly if their number is increased, but the results obtained are variable and rather imprecise4,5. Also, the technique does not allow isolation of clones, thus the mutational origin of thioguanine-resistant (TGr) cells cannot be determined as the gene product, HGPRT, cannot be measured. Previous methods for cloning peripheral blood lymphocytes (PBL) have been so inefficient that detection of TGr lymphocytes has not been practicable. We have cloned PBL by a technique which is highly efficient (20–60% of PBL form a clone) and results in large clones of 103–104 cells that are readily scored using an inverted microscope6,7. We report here that the in vivo mutation frequency is of the order of that observed for human fibroblast lines cultured in vitro1.

Different sensitivity to cytotoxic agents of internal and external cells of spheroids composed of thioguanine-resistant and sensitive cells.

When thioguanine-resistant V79 cells are introduced into spinner flasks containing V79 multicell spheroids, the mutant cells attach to the surface of the spheroids. The composite spheroids thus formed consist of external, thioguanine-resistant (TGr) and internal, thioguanine-sensitive cells. Cell sorting with a Becton Dickinson FACS II was used to determine the relative position of TGr cells and sensitivity to fluorescent drugs. After 2-4 days, the TGr cells are found internally as well as externally. The initial percentage of TGr cells varies from 1 to 50%, depending on the size of the single-cell inoculum, size of spheroids and frequency of addition of cells during a 24th period. Differential effects of drugs or radiation on external (cycling, oxic) vs internal (non-cycling, hypoxic) cells of composite spheroids can be assayed by simply plating cells trypsinized from spheroids into standard medium or medium with 2 mu g/ml 6-thioguanine, which allows growth of only TGr cells. For example, Adriamycin, which penetrates poorly into spheroids, is preferentially toxic to external cells, and causes a decrease in the percentage of TGr survivors. Irradiation of composite spheroids also causes preferential killing of external (oxic) TGr cells. However, AF-2, a nitrofuran which preferentially kills hypoxic cells, caused an increase in the 0/0 of TGr cells. This technique should prove useful in the evaluation of the preferential cytotoxicity of chemotherapeutic agents, alone or in combination.

Conditions for the selection of azaguanine- or thioguanine-resistant human diploid fibroblasts

The ability of posttreatment exposure to non-toxic concentrations of thymidine (TdR) to enhance the lethal effects of a number of alkylating agents, X-rays and UV and the lethal and mutagenic effects of N′-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) has been examined in V79 cell lines. TdR posttreatment enhanced the cytotoxic effects of ethyl methanesulphonate (EMS), MNU and ENU but not of UV or X-rays and increased both the spontaneous and MNU- and ENU-induced frequencies of azaguanine resistant (AZR) mutants. No significant effect of TdR on the spontaneous frequency of thioguanine resistant (TGR) mutants was demonstrated but the frequency of MNU-induced mutants to TGR was enhanced. The effects on expression of both potentially lethal and premutagenic damage were reversed by addition of an equimolar concentration of deoxycytidine (dCdR). The enhancement in spontaneous and induced mutant frequency (IMF) at the HGPRT locus appears to be due to an alteration in the selective efficiency of purine analogous due to alteration in growth kinetics of cells exposed to TdR or treated with alkylated agents or posttreated with thymidine after alkylation damage and not to an alteration in the miscoding potential of alkylated bases.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation, selection, and characterization of a mutant lacking hypoxanthine-guanine phosphoribosyltransferase activity by nutritional means.

Mutants of the Chinese hamster ovary cell derived from CHO-K1 have been selected for lack of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) without the use of a drug-resistance protocol. The procedure depends on the use of a parental strain carrying a mutation making it unable to synthetize purines and thus dependent upon exogenously added purines for growth. The standard "BUdR-visible-light" procedure is then used to select those cells which can use adenine but cannot use hypoxanthine as a purine source. These cells are shown to be thioguanine resistant, to be unable to incorporate exogenously added hypoxanthine into purine nucleotides, to complement our other adenine-specific purine auxotrophs, Ade-H and Ade-I but not to complement a cell isolated by virtue of thioguanine resistance, and to lack the activity of HGPRT. The use of such multiply marked mutants and cells related to them for further analysis of purine nucleotide biosynthesis and interconversion is discussed.