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Am J Translational Res 2012;4(1):24-43
Original Article
An atlas of histone deacetylase expression in breast cancer:
Fluorescence methodology for comparative semi-quantitative analysis
Katherine Ververis, Tom C Karagiannis
Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education
Precinct, Melbourne, Victoria, Australia; Department of Pathology, The University of Melbourne, Parkville, Victoria,
Australia.
Received December 7, 2011; accepted December 28, 2011; Epub January 5, 2011; Published January 15, 2011
Abstract: The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and
depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the
treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently
undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various
haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-
histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One
of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic
benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different
histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific
targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to
determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell
carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on
immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our
findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-
PCR and immunoblotting. Following validation of our approach we examined the expression of the different
isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of
class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer
cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating
potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be
utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of
histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that
such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors
become more readily available. (AJTR1112003).
Keywords: Chromatin, histone acetylation, histone deacetylase inhibitor, breast cancer, immunofluorescence
Full Text PDF
Address all correspondence to:
Dr Tom Karagiannis
Epigenomic Medicine
BakerIDI Heart and Diabetes Institute
75 Commercial Road, Melbourne, VIC, Australia
Phone: +613 8532 1309
Fax: +613 8532 1100
E-mail: tom.karagiannis@bakeridi.edu.au
Original Article
An atlas of histone deacetylase expression in breast cancer:
Fluorescence methodology for comparative semi-quantitative analysis
Katherine Ververis, Tom C Karagiannis
Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education
Precinct, Melbourne, Victoria, Australia; Department of Pathology, The University of Melbourne, Parkville, Victoria,
Australia.
Received December 7, 2011; accepted December 28, 2011; Epub January 5, 2011; Published January 15, 2011
Abstract: The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and
depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the
treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently
undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various
haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-
histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One
of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic
benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different
histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific
targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to
determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell
carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on
immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our
findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-
PCR and immunoblotting. Following validation of our approach we examined the expression of the different
isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of
class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer
cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating
potential complications with the use of class- or isoform-specific compounds. Overall, our approach can be
utilized to rapidly compare, in an unbiased semi-quantitative manner, the differential levels of expression of
histone deacetylase enzymes in cells and tissues using widely available imaging software. It is anticipated that
such analysis will become increasingly important as class- or isoform-specific histone deacetylase inhibitors
become more readily available. (AJTR1112003).
Keywords: Chromatin, histone acetylation, histone deacetylase inhibitor, breast cancer, immunofluorescence
Full Text PDF
Address all correspondence to:
Dr Tom Karagiannis
Epigenomic Medicine
BakerIDI Heart and Diabetes Institute
75 Commercial Road, Melbourne, VIC, Australia
Phone: +613 8532 1309
Fax: +613 8532 1100
E-mail: tom.karagiannis@bakeridi.edu.au
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