Abstract
BackgroundMetallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC).ResultsMT3 expression was not detected in one of the four oesophageal cell lines. The MT3 promoter was methylated in all of the oesophageal cell lines, but the degree of methylation was greater in the non-expressing cell line. After treatment with 5-aza-2'-deoxycytidine there was a reduction in the degree of methylation, and an increase in MT3 expression, in each of the cell lines (p < 0.01). Methylation was detected in 52% (33 of 64) of primary SCC and 3% (2 of 62) of histologically normal resection margins. MT3 expression was measured in 29 tumours, 17 of which had methylation of MT3. The expression of MT3 was significantly less in the methylated tumours compared to either the unmethylated tumours (p = 0.03), or the matched margin (p = 0.0005). There was not a significant difference in MT3 expression between the tumour and the margin from patients with unmethylated tumour. No correlations were observed between methylation of MT3 and survival time, patient age, gender, smoking or drinking history, tumour stage, volume, or lymph node involvement.ConclusionWe conclude that MT3 expression is frequently down-regulated in oesophageal SCC, by DNA methylation, but that this is not a prognostic indicator.
Highlights
Metallothionein 3 (MT3) inhibits growth in a variety of cell types
Methylation analysis of MT3 in normal lymphocytes and oesophageal cell lines The methylation status of the MT3 CpG island was measured by combined bisulfite restriction analysis (COBRA) within 3 overlapping regions (Figure 1)
There was no evidence of methylation at any of the other sites analysed by COBRA in the R1 and the R2 regions
Summary
Metallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC). Metallothionein 3 (MT3) was first identified as a growth inhibitory factor, expressed in normal brain, which inhibited the survival of neurones in culture and neurite formation [4]. Subsequent studies using glial [5] or tumour[6,7,8] cells, stably transfected with MT3, showed that its endogenous over-expression could inhibit cell growth. Methylation of the MT3 promoter has been observed and has been suggested to cause reduced expression in gastric cancer [11]
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