Elimination of non-viable 6-thioguanine-sensitive T cells from viable T cells prior to PCR analysis

Grant, Donna D.; Goldstein, Rose; Karsh, Jacob; Birnboim, H.C.

doi:10.1016/s0022-1759(99)00028-9

Cited by 2 publications

(3 citation statements)

References 13 publications

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“…Although the frequency of mutated T-cells is higher in RA and OA patients than that in controls, it was difficult to study the former at the DNA level because T-cells from these patients grew very slowly in culture, perhaps as a result of either the disease state or the medications. Many clones had fewer than 200 cells, the minimum number needed for reliable MP-PCR analysis [Grant et al, 1999].…”

Section: Discussionmentioning

confidence: 99%

“…TK6-D1 is an hprt negative derivative of the TK6 lymphoblastoid cell line, in which the entire hprt gene along with flanking anonymous markers DXS79 and DXS86 have been deleted. After 2 weeks, DNA was isolated from hprt mutant T-cell clones (approximately 200 live cells) for PCR analysis as reported elsewhere [Grant et al, 1999]. As a positive control, DNA was isolated from wild-type (i.e.…”

Section: Isolation Of Hprt Mutant T-lymphocyte Clones From Ra Oa Anmentioning

confidence: 99%

“…The only previous evidence of large deletions extending over one or more exons is indirect, coming from the study of mutant hypoxanthine phosphoribosyltransferase (hprt) mRNA [Zhuang et al, 1998[Zhuang et al, , 2000. To better understand the nature of mutations induced by RNOS, a human B-lymphoblastoid cell line (WIL2-NS) was exposed to RNOS and large deletions affecting the hprt gene were studied at the DNA level using multiplex-PCR [Grant et al, 1999].…”

Section: Introductionmentioning

confidence: 99%

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Nitric oxide donors induce large‐scale deletion mutations in human lymphoblastoid cells: Implications for mutations in T‐lymphocytes from arthritis patients

Grant

Goldstein

Karsh

et al. 2001

Environ and Mol Mutagen

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Rheumatoid arthritis (RA) is an inflammatory disease in which high levels of reactive nitrogen oxygen species (RNOS) may be present in the affected joints. RNOS are known to produce small-scale mutational events (transitions, transversions, small insertions, and small deletions) but the ability of these compounds to cause deletion of large segments of genomic DNA has not been previously determined. To address this question, a human lymphoblastoid cell line (WIL2-NS) was exposed to nitric oxide (NO)-donating drugs and hypoxanthine phosphoribosyltransferase (hprt)-negative clones were selected and analyzed by multiplex-PCR. Large-scale deletions accounted for 60-80% of hprt mutations arising in drug-treated cultures compared to 12% in untreated cultures (P-values of 0.006 and 0.0001, respectively, in two experiments). Deletion mutations in untreated cultures affected exon 9, whereas 75% of drug-induced deletion mutations affected exons 2, 3, and 9, and the remainder were very large, ranging from 26 to 1200 kbp. To compare this spectrum of NO-induced mutations in a lymphoblastoid line to that arising in vivo in arthritis patients, T-cells from RA patients, osteoarthritis (OA) patients, and controls were cloned and similarly analyzed. We previously showed that the overall frequency of Hprt mutant clones from patients is appreciably elevated compared to that of control subjects. Large-scale hprt deletions (0.5 to >26 kb) were detected in mutant T-cell clones from both RA and OA patients and also from control subjects. A total of 54 mutant clones from 16 RA patients and 19 mutant clones from 6 OA patients were studied. Of these, 6 clones (from 3 RA and 1 OA patient) had suffered large-scale deletions. A total of 9 control subjects were studied and 62 mutant clones were obtained. Of these, 19 had suffered large-scale deletions, arising in 7 of 9 control subjects. In conclusion, (1) RNOS are capable of inducing large-scale deletion mutations in a human lymphoblastoid cell line and (2) large-scale deletion mutations were found in 10-30% of T-cell clones from RA and OA patients and controls, which we hypothesize may be induced by RNOS.

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Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Hprt Mutant T-lymphocyte Clones From Ra Oa Anmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nitric oxide donors induce large‐scale deletion mutations in human lymphoblastoid cells: Implications for mutations in T‐lymphocytes from arthritis patients

Grant

Goldstein

Karsh

et al. 2001

Environ and Mol Mutagen

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Induction of mutant lymphocytes in cyclophosphamide- and chlorambucil-treated patients

Sanderson

Johnson²,

Henner³

2001

Mutagenesis

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Monitoring patients treated with single antineoplastic agents is aiding our understanding of what hazard these drugs pose in vivo. In this study, the frequency of mutant 6-thioguanine-resistant (TG(R)) peripheral blood lymphocytes was monitored before treatment and for < or =35 weeks after treatment of patients with cyclophosphamide (CP) or chlorambucil (CAB). The mean mutant frequency before treatment for six multiple sclerosis patients treated with high-dose CP was 2.53 x 10(-5) and increased after treatment to 4.61 x 10(-5) (P = 0.08, paired t-test). Using each patient as their own control, there were significant increases (each at P < 0.04) detectable within 2-4 weeks in four of the multiple sclerosis patients treated with CP. There was no increase in an untreated control monitored over the same period. In a patient receiving five sequential CP treatments at 1 month intervals, there were cumulative increases in the frequency of mutant cells. The mutant frequency increased from 0.31 x 10(-5) before treatment to 3.64 x 10(-5) after the final treatment and had decreased to 0.53 x 10(-5) at 35 weeks after treatment. In one of two CAB-treated patients with indolent non-Hodgkin's lymphoma, there was a significant increase in mutant frequency (P < 0.03) after treatment. Freshly isolated peripheral blood lymphocytes treated with 4-hydroperoxy-CP in vitro demonstrate a dose-dependent increase in mutant frequency. The increment in mutant frequency observed in vivo is of the order expected from the in vitro experiments. Although this study demonstrates that single or multiple doses of a single antineoplastic agent are mutagenic in vivo for some patients, further studies are needed to determine the extent and mechanism of the inter-individual variations in mutagenic response.

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Elimination of non-viable 6-thioguanine-sensitive T cells from viable T cells prior to PCR analysis

Cited by 2 publications

References 13 publications

Nitric oxide donors induce large‐scale deletion mutations in human lymphoblastoid cells: Implications for mutations in T‐lymphocytes from arthritis patients

Nitric oxide donors induce large‐scale deletion mutations in human lymphoblastoid cells: Implications for mutations in T‐lymphocytes from arthritis patients

Induction of mutant lymphocytes in cyclophosphamide- and chlorambucil-treated patients

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