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Molecular and epigenetic analysis of the fragile histidine triad tumour suppressor gene in equine sarcoids.

Strazzullo M, Corteggio A, Altamura G, Francioso R, Roperto F, D'Esposito M, Borzacchiello G - BMC Vet. Res. (2012)

Bottom Line: Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection.DNA methylation seems not to be involved in the gene expression alteration.Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Animal Health, University of Naples Federico II, Via Veterinaria, Naples, Italy.

ABSTRACT

Background: Sarcoids are peculiar equine benign tumours. Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection. Little is known about the molecular interplay between viral infection and neoplastic transformation. The data regarding papillomavirus infections in human species show the inactivation of a number of tumour suppressor genes as basic mechanism of transformation. In this study the putative role of the tumour suppressor gene Fragile Histidine Triad (FHIT) in sarcoid tumour was investigated in different experimental models. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissue.

Results: Nine paraffin embedded sarcoids and sarcoid derived cell lines were analysed for the expression of FHIT protein by immunohistochemistry, immunofluorescence techniques and western blotting. These analyses revealed the absence of signal in seven out of nine sarcoids. The two sarcoid derived cell lines too showed a reduced signal of the protein. To investigate the causes of the altered protein expression, the samples were analysed for the DNA methylation profile of the CpG island associated with the FHIT promoter. The analysis of the 32 CpGs encompassing the region of interest showed no significative differential methylation profile between pathological tissues and cell lines and their normal counterparts.

Conclusion: This study represent a further evidence of the role of a tumour suppressor gene in equine sarcoids and approaches the epigenetic regulation in this well known equine neoplasm. The data obtained in sarcoid tissues and sarcoid derived cell lines suggest that also in horse, as in humans, there is a possible involvement of the tumour suppressor FHIT gene in BPV induced tumours. DNA methylation seems not to be involved in the gene expression alteration. Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.

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Western blotting analysis of FHIT protein in sarcoid tissues and sarcoid derived cell lines. A) The expression of FHIT is reduced in sarcoids (S1, S2 and S3) compared to normal skin sample (N). B) FHIT protein expression in EqSO1a and EqSO4b in comparison with E-DERM. The lower blot shows actin expression to demonstrate the same amount of protein loaded on the gel.
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Figure 3: Western blotting analysis of FHIT protein in sarcoid tissues and sarcoid derived cell lines. A) The expression of FHIT is reduced in sarcoids (S1, S2 and S3) compared to normal skin sample (N). B) FHIT protein expression in EqSO1a and EqSO4b in comparison with E-DERM. The lower blot shows actin expression to demonstrate the same amount of protein loaded on the gel.

Mentions: We also analyzed three equine fibroblast cell lines for the expression of FHIT protein, one normal and two derived from sarcoids. By indirect immunofluorescence, distinct cytoplasmic staining was detected in the normal equine fibroblast cell line (E-DERM). The immunosignal was detected only within the cytosol and the pattern was diffuse. The sarcoid cell lines EqSO1a and EqSO4b showed very weak cytoplasmic immunofluorescence signal for FHIT protein as compared with E-DERM (Figure 2). However, EqSO4b cells showed a weaker immunofluorescence staining than EqSO1a. To further confirm the lack of FHIT protein expression in tumours, three sarcoid samples, one skin sample from a healthy horse and the cell lines were analysed by immunoblotting. The anti-FHIT antibody recognized a band of the expected molecular weight in the neoplastic tissues, normal skin and all fibroblast cell lines. An increase in the amount of FHIT protein level in non-neoplastic skin versus tumour samples was observed (Figure 3A); moreover FHIT expression levels were lower in sarcoid cell lines compared to E-DERM (Figure 3B).


Molecular and epigenetic analysis of the fragile histidine triad tumour suppressor gene in equine sarcoids.

Strazzullo M, Corteggio A, Altamura G, Francioso R, Roperto F, D'Esposito M, Borzacchiello G - BMC Vet. Res. (2012)

Western blotting analysis of FHIT protein in sarcoid tissues and sarcoid derived cell lines. A) The expression of FHIT is reduced in sarcoids (S1, S2 and S3) compared to normal skin sample (N). B) FHIT protein expression in EqSO1a and EqSO4b in comparison with E-DERM. The lower blot shows actin expression to demonstrate the same amount of protein loaded on the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3361464&req=5

Figure 3: Western blotting analysis of FHIT protein in sarcoid tissues and sarcoid derived cell lines. A) The expression of FHIT is reduced in sarcoids (S1, S2 and S3) compared to normal skin sample (N). B) FHIT protein expression in EqSO1a and EqSO4b in comparison with E-DERM. The lower blot shows actin expression to demonstrate the same amount of protein loaded on the gel.
Mentions: We also analyzed three equine fibroblast cell lines for the expression of FHIT protein, one normal and two derived from sarcoids. By indirect immunofluorescence, distinct cytoplasmic staining was detected in the normal equine fibroblast cell line (E-DERM). The immunosignal was detected only within the cytosol and the pattern was diffuse. The sarcoid cell lines EqSO1a and EqSO4b showed very weak cytoplasmic immunofluorescence signal for FHIT protein as compared with E-DERM (Figure 2). However, EqSO4b cells showed a weaker immunofluorescence staining than EqSO1a. To further confirm the lack of FHIT protein expression in tumours, three sarcoid samples, one skin sample from a healthy horse and the cell lines were analysed by immunoblotting. The anti-FHIT antibody recognized a band of the expected molecular weight in the neoplastic tissues, normal skin and all fibroblast cell lines. An increase in the amount of FHIT protein level in non-neoplastic skin versus tumour samples was observed (Figure 3A); moreover FHIT expression levels were lower in sarcoid cell lines compared to E-DERM (Figure 3B).

Bottom Line: Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection.DNA methylation seems not to be involved in the gene expression alteration.Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Animal Health, University of Naples Federico II, Via Veterinaria, Naples, Italy.

ABSTRACT

Background: Sarcoids are peculiar equine benign tumours. Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection. Little is known about the molecular interplay between viral infection and neoplastic transformation. The data regarding papillomavirus infections in human species show the inactivation of a number of tumour suppressor genes as basic mechanism of transformation. In this study the putative role of the tumour suppressor gene Fragile Histidine Triad (FHIT) in sarcoid tumour was investigated in different experimental models. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissue.

Results: Nine paraffin embedded sarcoids and sarcoid derived cell lines were analysed for the expression of FHIT protein by immunohistochemistry, immunofluorescence techniques and western blotting. These analyses revealed the absence of signal in seven out of nine sarcoids. The two sarcoid derived cell lines too showed a reduced signal of the protein. To investigate the causes of the altered protein expression, the samples were analysed for the DNA methylation profile of the CpG island associated with the FHIT promoter. The analysis of the 32 CpGs encompassing the region of interest showed no significative differential methylation profile between pathological tissues and cell lines and their normal counterparts.

Conclusion: This study represent a further evidence of the role of a tumour suppressor gene in equine sarcoids and approaches the epigenetic regulation in this well known equine neoplasm. The data obtained in sarcoid tissues and sarcoid derived cell lines suggest that also in horse, as in humans, there is a possible involvement of the tumour suppressor FHIT gene in BPV induced tumours. DNA methylation seems not to be involved in the gene expression alteration. Further studies are needed to understand the basic molecular mechanisms involved in reduced FHIT expression.

Show MeSH
Related in: MedlinePlus