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Understanding the Mechanisms of FHIT Inactivation in Cervical Cancer for Biomarker Development

Raheela Ashfaq, MD

Sabeeha Muneer, PhD

David G. Burbee, PhD

David S. Miller, MD

John D. Minna, MD

Departments of Obstetrics and Gynecology and Pathology, and Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas

Carolyn Y. Muller, MD

Departments of Obstetrics and Gynecology and Pathology, and Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas; carolyn.muller{at}utsouthwestern.edu

Objective: Loss of the fragile histidine triad (Fhit) protein has been documented in cervical cancer and dysplasia. The goal of this study was to confirm the utility of homozygous deletions, aberrant methylation, and immunohistochemical evaluations of FHIT as functionally relevant determinants of FHIT expression.

Methods: We studied matched DNA, RNA, and protein from nine early-passage cervical cancer cell lines. DNA markers spanning FHIT were used to examine the extent of homozygous deletions for each cell line. 5' CpG island methylation of FHIT was investigated by methylation-specific polymerase chain reaction (PCR) assays. FHIT transcripts were characterized by reverse transriptase (RT)-PCR. Western blot analysis and immunohistochemistry were performed to characterize Fhit protein expression.

Results: Homozygous deletions were found in six of nine cervical cancer cell lines, but only had homozygous deletions involving an exon. All nine lines had both methylated and unmethylated alleles according to methylation-specific PCR. Loss of wild-type FHIT transcripts were found in five of nine lines. By western blot analysis, Fhit protein expression was lost in five of nine lines, producing an exact correlation with RT-PCR results. Immunohistochemical staining was concordant with Fhit protein expression by western blotting in eight of nine cell lines.

Conclusion: A perfect correlation was found between FHIT mRNA expression and western blot analysis. Assays for Fhit protein expression and large FHIT homozygous deletions are representative biomarkers of Fhit expression. By contrast, the aberrant methylation assay is not concordant with FHIT gene expression, and we suggest caution in its use as a functionally relevant biomarker for cervical cancer.

Key Words: FHIT • cervical cancer • aberrant methylation

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