Role of 5'-CpG island hypermethylation of the FHIT gene in cervical carcinoma

Abstract

Objective: The abnormal expression of fragile histidine triad (FHIT) gene has been frequently reported in a variety of epithelial malignancies including cervical carcinoma. Furthermore, in a recent study it was proposed that transcriptional inactivation of FHIT, as a consequence of aberrant 5'-CpG island methylation, plays an important role in the carcinogenesis of human cervical carcinoma. The authors sought to determine whether abnormal FHIT transcription occurs in human cervical carcinoma, and if so, whether this abnormal expression is associated with aberrant 5'-CpG island methylation. In addition, the clinical significance of FHIT inactivation was investigated in Korean women with cervical cancer.

Methods: To examine for abnormal transcripts of the FHIT gene, quantitative RT-PCR, genomic DNA-PCR and nonisotopic RT-PCR-SSCP analysis were performed using the standard method. The methylation status was determined by methylation specific PCR and bisulfite DNA sequencing.

Results: The FHIT gene was down-regulated in 15 of 58 (25.9%) cervical carcinomas. FHIT promoter hypermethylation was detected in 15 of 15 (100%) abnormally expression in cervical carcinomas. Bisulfite DNA sequencing confirmed these findings and a significant correlation was found between CpG site hypermethylation and low FHIT expression. However, no significant correlation was found between reduced FHIT expression and clinicopathological characteristics.

Conclusion: In this study, FHIT inactivation in cervical cancer was found to be strongly correlated with 5'-CpG island hypermethylation rather than a genetic alteration. Furthermore, no significant relation was found between a lack of FHIT expression and the prognostic factors of cervical cancer in our Korean cohort.

Keywords: Bisulfite-DNA sequencing; Cervical carcinoma; FHIT gene; Hypermethylation.

Fig. 1

Reduced expression of FHIT mRNA in human cervical cancer tissues (quantitative RT-PCR analysis). N; normal cervix tissue, T; cervical cancer tissue.

Fig. 2

FHIT 5'-CpG island hypermethylation in human cervical cancer (methylation-specific PCR analysis). N; normal cervix tissue, T; cervical cancer tissue, U; unmethylation-specific PCR, M; methylation-specific PCR.

Fig. 3

FHIT 5'-CpG island hypermethylation in human cervical cancer (sodium bisulfite DNA sequencing analysis). PCR products were cloned, and 5 plasmid clones were sequenced (■ methylated CpG; □ unmethylated CpG). methylation status of 13 CpGs in FHIT promoter. mean methylation status was determined from the number of alleles containing a methylated CpG at each position (■ complete methylation; 4-5 clones, partial methylation; 2-3 clones, □ unmethylation; 0-1 clone).

Fig. 4

Methylation status of the FHIT CpG Island in cervical cancer (correlation between CpG site hypermethylation with low FHIT expression). N; normal cervical tissue, T; cervical cancer tissue.