Evidence for Functionally Significant Polymorphism of Human Glutamate Cysteine Ligase Catalytic Subunit: Association with Glutathione Levels and Drug Resistance in the National Cancer Institute Tumor Cell Line Panel

Abstract

Glutamate-cysteine ligase (GCL) is the first and rate-limiting enzyme involved in the biosynthesis of glutathione (GSH). The GCL heterodimer is encoded by two genes: GLCLC, which directs synthesis of the catalytic subunit, and GLCLR, which encodes the regulatory subunit. We have previously identified a polymorphic GAG/CTC trinucleotide repeat within the 5' untranslated region of GLCLC. Here we report the further characterization of GLCLC polymorphism and the existence of five GLCLC alleles as defined by the trinucleotide repeat, which exhibits a range of 4 to 10 uninterrupted repeats. Significant variation in GLCLC allele frequencies was observed in four different ethnic populations examined. Interindividual variation in the capacity to produce GSH due to GLCLC polymorphism is hypothesized to influence the cellular response to environmental toxicants and chemotherapeutic agents. To test this hypothesis, the 60 tumor cell lines of the National Cancer Institute drug screening panel were genotyped for the GLCLC trinucleotide repeat, and the association of GLCLC genotype with GSH levels and drug sensitivity/resistance data was examined. Here we demonstrate an association between certain GLCLC alleles and GSH levels and/or drug sensitivity, providing evidence that suggests polymorphism of human GLCLC is functionally significant.

Variants in the Glutamate-Cysteine-Ligase Gene Are Associated with Cystic Fibrosis Lung Disease

“…Mean FEV 1 (% predicted) was 62% (range, 11-128%). GCLC GAG TNR genotype frequencies are shown in Table 2 and were similar to that observed in the white population (20). GCLC GAG TNR genotype frequencies were in Hardy-Weinberg equilibrium.…”

“…Second, we assumed a linear dose-response relationship between GCLC GAG TNR genotype and CF lung function. This is based on earlier data that suggest that the GCLC 7 GAG TNR polymorphisms result in less GSH compared with the 8 GAG TNR and 9 GAG TNR (20). This has yet to be confirmed in patients with CF.…”

“…The 5Ј-untranslated region of GCLC mRNA (NM_001498) has been shown to contain a polymorphic guanine-adenine-guanine (GAG) trinucleotide repeat (TNR) just upstream of the translation start codon (19). This GAG TNR polymorphism is associated with variable levels of GSH in human cells lines, with 7 GAG TNR associated with the lowest GSH levels, 8 GAG TNR associated with intermediate GSH levels, and 9 GAG TNR associated with the highest GSH levels (20).…”

Regulation of glutathione synthesis

“…Polymorphism of GCL subunits has received increasing attention. GCLC polymorphism has been shown to be associated with decreased GCLC expression in a patient with hemolytic anemia (Beutler et al, 1999), GSH levels and drug resistance among tumor cell lines (Walsh et al, 2001), decreased GCL activity and GSH levels in patients with schizophrenia (Gysin et al, 2007). GCLM polymorphism has been associated with impairment of NO-mediated coronary vasomotor function and risk of myocardial infarction (Nakamura et al, 2002;Nakamura et al, 2003).…”

Functional significance of the GAG trinucleotide-repeat polymorphism in the gene for the catalytic subunit of γ-glutamylcysteine ligase

“…Polymorphisms have been identified in both the GCLC and GCLM genes, some of which have been associated with decreased GSH levels in vitro and altered susceptibility to certain diseases [15][16][17]. A trinucleotide (GAG) tandem repeat polymorphism, identified in the 5′ untranslated region (exon 1, 10 base pairs upstream of the ATG start site) of GCLC has been identified with alleles consisting of 4, 7, 8, 9 and 10 consecutive GAG repeats; 7 and 9 being most prevalent [18]. It has been observed that the distribution of the allele with the 4 GAG-repeat is restricted to blacks and the 10 GAG-repeat allele restricted to blacks and Hispanics [18].…”