Line-1 hypomethylation in multistage carcinogenesis of the uterine cervix.

OBJECTIVE: To evaluate characteristics of global hypomethylation in evolution of cervical cancer. MATERIALS AND METHODS: Eight cases of squamous cell carcinoma (SCC) and seven cases of carcinoma in situ (CIS) were studied. Each of the SCC samples contained CIS, and all SCC and CIS samples contained normal ectocervical epithelium. Microdissection was performed to separate normal epithelium, CIS and SCC prior to DNA extraction. Hypomethylation levels of long interspersed nuclear elements (LINE-1 or L1) were measured with a combined bisulfite restriction analysis (COBRA) PCR (polymerase chain reaction) protocol. The percentage of L1 hypomethylation for SCC, CIS and normal epithelium was compared. RESULTS: In the SCC cohort, the L1 hypomethylation level showed progressive increase comparing normal epithelium (59.4 +/- 8.86%) to CIS (64.37 +/- 7.32%) and SCC (66.3 +/- 7.26%) (repeated measurement ANOVA, P = 0.005). A significantly greater L1 hypomethylation level was found in CIS (62.06 +/- 3.44 %) compared to normal epithelium (60.03 +/- 3.69 %) (paired t-Test, P = 0.03). No significant difference in L1 hypomethylation level was noted between CIS of the two sample groups (unpaired t-Test, P = 0.2). CONCLUSIONS: In our study, there was a significant correlation between the degree of hypomethylation and progression from normal ectocervical mucosa to CIS and invasive cancer. Laboratory assessment of biopsies for this molecular event may have clinical significance.

Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma.

BACKGROUND: We investigated the clinical implications of global hypomethylation, one of the most consistent epigenetic changes in cancer, in the sera of patients with hepatocellular carcinoma (HCC). METHODS: Combined bisulfite restriction analysis PCR was used to assess the methylation status of LINE-1 repetitive sequences in genomic DNA derived from sera of 85 patients with HCC, 73 patients with cirrhosis, 20 healthy carriers of hepatitis B virus (HBV) and 30 healthy controls. RESULTS: Serum genome hypomethylation, the percentage of unmethylated LINE-1, was significantly increased in patients with HCC (P<0.001). The levels of serum LINE-1 hypomethylation at initial presentation correlated significantly with the presence of HBsAg, large tumor sizes, and advanced tumor stages classified by the CLIP score. Multivariate analyses showed that serum LINE-1 hypomethylation was a significant and independent prognostic factor of overall survival. CONCLUSION: Serum LINE-1 hypomethylation may serve as a prognostic marker for patients with HCC.

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis.

Genome-wide losses of DNA methylation have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. Limited information is available on the global methylation status in normal tissues and other cancer types beyond colonic carcinoma. Here we applied the combined bisulfite restriction analysis PCR to evaluate the methylation status of LINE-1 repetitive sequences in genomic DNA derived from microdissected samples from several human normal and neoplastic tissues. We found that methylation of LINE-1 in leukocytes was independent of age and gender. In contrast, normal tissues from different organs showed tissue-specific levels of methylated LINE-1. Globally, most carcinomas including breast, colon, lung, head and neck, bladder, esophagus, liver, prostate, and stomach, revealed a greater percentage of hypomethylation than their normal tissue counterparts. Furthermore, DNA derived from sera of patients with carcinoma displayed more LINE-1 hypomethylation than those of noncarcinoma individuals. Finally, in a colonic carcinogenesis model, we detected significantly greater hypomethylation in carcinoma than those of dysplastic polyp and histological normal colonic epithelium. Thus, the methylation status is a unique feature of a specific tissue type and the global hypomethylation is a common epigenetic process in cancer, which may progressively evolve during multistage carcinogenesis.