title: Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development.
abstract: 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 transcriptase (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 one 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9032, USA.
authors.forename: Jayanthi S
authors.initials: JS
authors.lastname: Lea
authors.forename: Raheela
authors.initials: R
authors.lastname: Ashfaq
authors.forename: Sabeeha
authors.initials: S
authors.lastname: Muneer
authors.forename: David G
authors.initials: DG
authors.lastname: Burbee
authors.forename: David S
authors.initials: DS
authors.lastname: Miller
authors.forename: John D
authors.initials: JD
authors.lastname: Minna
authors.forename: Carolyn Y
authors.initials: CY
authors.lastname: Muller
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics*; D001483:Base Sequence; D015415:Biomarkers; D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics*; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic; D002583:Uterine Cervical Neoplasms / Q000235:genetics*; D000099170:Bis(5'-adenosyl)triphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D015415:Biomarkers; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000099170:Bis(5'-adenosyl)triphosphatase; C015772:CpG dinucleotide