Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development

Jayanthi S. Lea, Raheela Ashfaq, Sabeeha Muneer, David G. Burbee, David S. Miller, John D. Minna, Carolyn Y. Muller

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 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 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. 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.

Original languageEnglish (US)
Pages (from-to)329-337
Number of pages9
JournalJournal of the Society for Gynecologic Investigation
Volume11
Issue number5
DOIs
StatePublished - Jul 2004

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Tumor Biomarkers
Uterine Cervical Neoplasms
Methylation
Western Blotting
Cell Line
Polymerase Chain Reaction
Biomarkers
Uterine Cervical Dysplasia
CpG Islands
Genetic Markers
Histidine
Exons
Immunohistochemistry
Alleles
fragile histidine triad protein
RNA
Staining and Labeling
Gene Expression
Messenger RNA
DNA

Keywords

  • aberrant methylation
  • FHIT, cervical cancer

ASJC Scopus subject areas

  • Obstetrics and Gynecology

Cite this

Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development. / Lea, Jayanthi S.; Ashfaq, Raheela; Muneer, Sabeeha; Burbee, David G.; Miller, David S.; Minna, John D.; Muller, Carolyn Y.

In: Journal of the Society for Gynecologic Investigation, Vol. 11, No. 5, 07.2004, p. 329-337.

Research output: Contribution to journalArticle

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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 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 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. 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.",
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N2 - 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 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. 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.

AB - 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 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. 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.

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