Expression of heavy subunit of γ-glutamylcysteine synthetase (γ-GCSh) in human colorectal carcinoma

Gamma-glutamylcysteine synthetase (γ-GCS) is a heterodimer consisting of heavy (γ-GCSh) and light (γ-GCS1) sub-units. γ-GCS catalyzes the rate-limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress. Expression of γ-GCSh and γ-GCS1 are sensitive to oxidative stress. To investigate whether expression of γ-GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients. In adjacent normal colorectal epithelium, levels of γ-GCSh expression were low. Strong cytoplasmic staining for γ-GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas. The frequency of γ-GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001). We used RNase protation assay and Western blot to determine levels of γ-GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls. Elevated expression of both γ-GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or post-transcriptional regulation play an important role in the upregulation of γ-GCS during colorectal carcinogenesis. We also examined the expression of another redox-regulated gene, multidrug resistance protein I (MRP1). Strong staining for MRP1 was detected in I (6.3%) adenoma and 40 (70.2%) carcinomas. The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma (p<0.0001). Nuclear p53 expression was detected in 30 (52.6%) of carcinomas. There is a significant correlation between γ-GCSh and MRP1 expression (p=0.013) but not between γ-GCSh and p53. Since γ-GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression.