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Doi:10.1016/j.mcp.2005.09.008Molecular and Cellular Probes 20 (2006) 81–86 Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) Department of Pathology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA Received 19 May 2005; accepted for publication 29 September 2005 In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and b actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states andcell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications andDNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probedthrough the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeuticstrategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the mostwidely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performedquantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D andZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, b actin, GAPDH and ribosomal highly-basic23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA,b actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment.
Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, b actin mRNA and GAPDH mRNA levels variedsignificantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as astandard for normalization during TSA treatment.
q 2006 Elsevier Ltd. All rights reserved.
Keywords: Housekeeping genes; Histone deacetylase inhibitor (HDACi); Trichostatin A (TSA); Ribosomal highly basic 23-kDa protein (rb 23-kDa; RPL13A);Quantitative RT-PCR; Epigenetic; Normalization.
a glycolytic enzyme, encoded by a single gene and it has theadvantage of being highly conserved in different species .
Assessment of gene expression by RT-PCR is based on b actin mRNA and 18S rRNA are constitutively expressed in comparison with internal standards, so called housekeeping eukaryotic cells and their expression is thought to remain genes. The use of such genes as internal control relies on the constant even during cell growth and during different phases of fact that ideally they should exhibit a constant basal level of the cell cycle . However, some studies have indicated expression which is consistent, non-regulated and independent increased expression of GAPDH and b actin mRNAs in of the cell cycle . 18S ribosomal RNA (rRNA), glyceral- carcinomas, in regenerating tissues after resection and during dehyde-3-phosphate dehydrogenase (GAPDH) messenger postnatal development Therefore, 18S rRNA is widely RNA (mRNA) and b actin mRNA are commonly used as used as a housekeeping gene. However, no one single such internal standards. However, it has now become clear that housekeeping gene is perfect under all experimental conditions the expression of these genes can be affected by various factors and therefore, it is necessary to characterize the suitability of like developmental and differentiation status, cell-cycle phase, various housekeeping genes to serve as internal controls under pathological conditions and drug treatments . GAPDH is An emerging area of research in cancer involves the study of epigenetic aberrations and their contribution to malignant transformation and progression of cancer. DNA methylation E-mail address: [email protected] (S.A. Abdulkadir).
and histone modifications (such as acetylation, methylation, 0890-8508/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
phosphorylation and ubiquitination) by specific chromatin- A. Mogal, S.A. Abdulkadir / Molecular and Cellular Probes 20 (2006) 81–86 modifying enzymes play essential roles in both tumor initiation tissue was treated with either vehicle (Ethanol) or Trichostatin and progression Among all of these histone modifications A (TSA, Sigma) at an optimal concentration of 500 ng/ml for acetylation is arguably the best-studied modification. The 8 h. Tissue was harvested in DMEM/F12 50/50 media actual levels of acetylation of the core histones result from supplemented with 5% FBS at 37 8C with 5% CO2. To our steady state balance between the opposing activities of histone knowledge, conditions for TSA treatment of mouse prostate acetyl-transferases (HATs) and histone deacetylases (HDACs) explants have not been determined. Therefore, we first Over the years many different types of HDAC inhibitors determined the effects of TSA treatment on histone acetylation (HDACi) have been developed, ranging from complicated in mouse prostate tissue explants using different concentrations structures of bacterial or fungal origin [trichostatin A (TSA), of TSA (100 ng/ml, 200 ng/ml and 500 ng/ml) and at different trapoxin] to the very simple butyrate. HDACi are capable of time points (8, 16 and 24 h) and found 500 ng/ml TSA for 8 h inhibiting HDACs with varying efficiencies (at nanomolar to to be the optimal condition for inducing histone H3 acetylation.
millimolar concentrations) leading to hyperacetylation ofhistones followed by transcriptional activation of certain genes TSA (at nanomolar concentrations) is the mostcommonly used HDACi. Furthermore, modulation of histone Human prostate cancer cell line (LNCaP) and human breast acetylation is currently being explored as a therapeutic strategy cancer cell lines (T-47D and ZR-75-1) were obtained from the in treatment of cancer. Specifically, inhibition of histone American Type Tissue Culture Collection (Manassas, Virgi- deacetylases by trichostatin A (TSA) has been shown to nia). Cell lines were cultured routinely in RPMI 1640 prevent tumorigenesis and metastasis We have been supplemented with 5% FBS at 37 8C with 5% CO2. To test interested in studying the role of chromatin state and histone the expression of internal standards, cells with fresh media acetylation on stochastic, dosage-sensitive gene regulation by were treated with either a vehicle (Ethanol) or Trichostatin A Nkx3.1 in prostate cancer. Nkx3.1 is a homeodomain-contain- (TSA, Sigma) at two different concentrations (100 ng/ml and ing transcription factor and candidate tumor suppressor gene whose deletion in mice leads to the formation of prostaticintraepithelial neoplasia (PIN) Loss of Nkx3.1 protein 2.4. RNA isolation and quantitative RT-PCR analysis expression is common in human prostate carcinomas andprostatic intraepithelial neoplasia (PIN) lesions and correlates RNA was extracted with Trizol reagent (Invitrogen) after 8 with tumor progression. Further, in the prostate, tumor and 24 h treatments from mouse prostate tissue and human initiation is often linked to loss of heterozygosity at the prostate and breast cancer cells respectively. Samples were Nkx3.1 locus and microarray analysis has identified Nkx3.1 dissolved in RNase-free water and quantified by spectro- target genes, some of which show exquisite dosage sensitivity photometric readings at 260 nm (A260). Purity of total RNA The number of Nkx3.1 alleles determines the relative was determined by the A260/A280 and A260/A230 ratio, and then probabilities of stochastic activation or inactivation of a given integrity of RNA samples was confirmed by electrophoresis on target gene In order to study the stochastic and dosage- 1% agarose gels. 1 mg of total RNA was reverse transcribed sensitive expression of these genes we needed an accurate using primer cocktail (200 ng/ml oligodT and 50 ng/ml of internal standard following TSA treatment. Here we report the random hexamer). Reaction mix contained 10 mM dNTP’s, use of ribosomal highly basic 23-kDa protein (rb 23-kDa, 0.1 M DTT, RNAsin and M-MLV reverse transcriptase RPL13A) as an adequate internal standard following TSA (Gibco/BRL 200 units/ml). Reaction conditions were 68 8C for 10 min and 42 8C for 60 min. RNase free water was used tomake final volume of 250 ml. cDNA samples were then boiled for 5 min and stored at K20 80C. PCR was performed bySYBRw green PCR Master Mix (Applied Biosystems).The increase in fluorescence of the SYBR green dye was monitoredusing a GeneAmp 5700 sequence detection system (Applied Trichostatin A was obtained (Sigma biochemical) and Biosystems). All of the PCR reactions were performed in dissolved into ethanol. Several aliquots of stock solutions of triplicate and independently repeated at least two to three 500 mg/ml, 200 mg/ml and 100 mg/ml were prepared and stored at K208C. Fresh aliquots were used for separate experiments.
2.2. Mouse prostate explants and TSA treatment Mouse prostate explants were treated with TSA as described A total of nine (nZ9) C57BL/6 male mice (Jackson Lab) in Section 2.2 and lysates were prepared using extraction buffer ranging between ages 12 weeks to 15 weeks were used. Three (50 mM Tris–HCl-buffered saline, pH 7.4, 1% Triton X-100, different experiments with three mice each were done under the 1% Nonidet P-40, 5 mM CaCl2, 2 mM phenylmethylsulfonyl same conditions. Prostates were extracted, divided into two fluoride, and 3 mM hydrogen peroxide). Nuclear protein was halves (1/2 for vehicle and 1/2 for TSA treatment) and further extracted by the method of Dignam et al Protein minced on culture plates under aseptic conditions. Prostate concentrations were measured using the Bio-Rad DC protein A. Mogal, S.A. Abdulkadir / Molecular and Cellular Probes 20 (2006) 81–86 F: 50-ACCAGTTCGCCATGGATGAC-30R: 50-TGCCGGAGCCGTTGTC-30 F: 50-CCAGCTCACCATGGATGATG-30R: 50-ATGCCGGAGCCGTTGTC-30 Fig. 1. Western blot analysis for acetylated and total histone H3 using mouse prostate explants. Mouse prostate explants were treated with either a vehicle (ethanol) or trichostatin A (TSA) at a concentration of 500 ng/ml for 8 h. Proteins were extracted, electrophoresed and blotted for acetylated H3 and total histone H3 levels. (A) Effect of trichostatin A (TSA) on the levels of total histone H3, (B) Effect of trichostatin A (TSA) on the levels of acetylated histone H3.
F: 50-CCCATGTTCGTCATGGGTGT-30R: 50-TGGTCATGAGTCCTTCCACGATA-30 H3 in TSA treated samples as compared to vehicle (ethanol) assay reagent. Extracts containing 20–30 mg of protein were treated samples (Our western blot findings confirmed electrophoresed on a 12% SDS-polyacrylamide gel and blotted that trichostatin A (TSA) treatment was effective in our membrane was blocked with 5% fat-free dry milk for 1 h atroom temperature and incubated with the rabbit polyclonal 3.2. RT-PCR quantitation of housekeeping genes following acetylated histone 3 antibody (1:1000, Upstate) at 4 8C trichostatin A (TSA) treatment in mouse prostate tissue and overnight. The membrane was then incubated for 1 h at room temperature with a peroxidase-labeled goat anti-rabbit anti-body (1:3000, Bio-Rad). The membrane was rinsed, treated In mouse prostate tissue explants, 18S rRNA (P!0.002) with ECL reagent (PerkinElmer Life Sciences) for 1 min and and b actin mRNA (p!0.01) levels were significantly reduced exposed to X-ray film at room temperature for 30 s to 1 min.
in TSA treated tissue samples as compared to the vehicle Membranes were stripped and then incubated with rabbit treated samples However, RPL13A and GAPDH polyclonal total histone three antibody (1:1000, Upstate).
mRNA levels remained unaffected and showed constantexpression in both the vehicle as well as TSA treated samples.
We extended these findings by examining the expression of these housekeeping genes in the human prostate cancer cell Primers were designed using the Primer express software line (LNCaP). In LNCaP cells, at a concentration of 100 ng/ml (ABI Prism) in our laboratory and synthesized by Integrated TSA, 18S rRNA (P!0.002) and b actin mRNA (p!0.01) DNA Technologies, Inc (Iowa). The sequences of all primers levels were significantly up-regulated in TSA treated samples (A). RPL13A and GAPDH mRNA levels remainedunaffected with TSA treatment. Further in LNCaP cells, at 2.7. Quantitation and statistical analysis 200 ng/ml TSA, GAPDH mRNA levels were most significantlyreduced (P!0.001) followed by 18S rRNA levels (P!0.01) Results of qRT-PCR for each gene are presented relative to the expression in the vehicle treated sample, which is set as100%. The difference in Ct value was used to calculatedifference between vehicle and TSA treated sample. Resultsare shown as Mean G SD and significance was calculated byusing paired t-test. Differences were considered significant atP%0.05.
3.1. Effects of TSA on histone acetylation in mouse prostate Fig. 2. Quantitative RT-PCR analysis of housekeeping genes following trichostatin A (TSA) treatment in mouse prostate tissue. 18S rRNA (P!0.002)and b actin mRNA (P!0.01) levels were significantly down-regulated in TSAtreated tissue samples as compared to the vehicle treated samples. However, Mouse prostate explants were treated with TSA (500 ng/ml) RPL13A and GAPDH mRNA levels remained unaffected. The data shown here or vehicle for 8 h and tissue extracts were used for western blot represent the average of three different experiments performed in triplicate analysis. We observed increased levels of acetylated histone using the same conditions. (**P!0.005, *P!0.05).
A. Mogal, S.A. Abdulkadir / Molecular and Cellular Probes 20 (2006) 81–86 The expression of housekeeping genes such as 18S rRNA, b actin mRNA and GAPDH mRNA ideally should remainconstant under all experimental conditions, in all normal aswell as pathological states and during cell growth and variousphases of the cell cycle However, some reports suggestedthat these genes may either be up-regulated or down-regulateddepending on the circumstances To explore whetherexpression of these housekeeping genes is modulated under theeffect of the commonly used histone deacetylase inhibitor(HDACi) trichostatin A (TSA), we compared the expressionlevel of these housekeeping genes (18S rRNA, b actin andGAPDH) along with the novel ribosomal highly basic 23-kDaprotein (rb 23-kDa, RPL13A). We examined the expressionpattern of all four housekeeping genes in mouse prostate tissue,human prostate cancer (LNCaP) cell line and human breastcancer (T-47D and ZR-75-1) cell lines following vehicle Fig. 3. Quantitative RT-PCR analysis of housekeeping genes following (Ethanol) or trichostatin A (TSA) treatment. To our knowl- trichostatin A (TSA) treatment in human prostate cancer (LNCaP) cell line. (A) edge, this is the first report of testing housekeeping genes as LNCaP (100 ng/ml TSA): 18S rRNA (P!0.002) and b actin mRNA (P!0.01) RNA internal standards under the effect of trichostatin A levels were significantly up-regulated in TSA treated samples as compared to the vehicle treated samples. However, RPL13A and GAPDH mRNA levelsremained unaffected. (B) LNCaP (200 ng/ml TSA): GAPDH mRNA (P! Our results showed that the commonly used housekeeping 0.001) and 18S rRNA (P!0.01) levels were significantly down-regulated in genes such as 18S rRNA, b actin mRNA, GAPDH mRNA TSA treated samples as compared to the vehicle treated samples. However, levels were significantly altered in the TSA treated samples as RPL13A and mRNA levels remained unaffected. The data show the average of compared to the vehicle treated samples. It is interesting that two different experiments performed in triplicate using the same conditions.
TSA can either up-regulate (e.g. LNCaP and ZR-75-1 at 100 ng/ml TSA) or down-regulate (e.g. prostate explants,T-47D, LNCaP and ZR-75-1 at 200 ng/ml TSA) expression of and b actin mRNA levels (B). However, RPL13A mRNA these genes. This could be due to an ‘optimal dose response levels remained unaffected at higher concentrations of TSA effect’ of TSA (100 ng/ml vs 200 ng/ml) in the particular tissue treatment as well and showed constant mRNA level or cell line. Regardless of the underlying reason, these findings underscore the importance of testing the suitability ofhousekeeping genes in different experimental systems.
The levels of ribosomal RNA, which make up 3.3. RT-PCR quantitation of housekeeping genes following total RNA, are thought to be less likely to vary under trichostatin A (TSA) treatment in human breast cancer conditions that affect the expression of mRNAs, since they are transcribed by a distinct RNA polymerase. 18S rRNA hasbeen described as a preferable internal control and most widely To extend our investigation in non-prostate cells we used used as a housekeeping gene . It is expressed at constant two different human breast cancer cell lines (T-47D and ZR- levels in normal liver versus liver metastasis . It is also 75-1). In T-47D cells, at 100 ng/ml TSA, 18S rRNA (P! stably expressed in various cancer tissues which may result 0.005), b actin mRNA (p!0.003) and GAPDH mRNA (P! from its lack of involvement in cellular metabolism .
0.01) levels were significantly reduced in TSA treated samples However, other studies have shown that 18S rRNA is not a RPL13A mRNA levels remained unaffected. Further suitable control as it can be regulated and its synthesis is at a concentration of 200 ng/ml TSA, b actin mRNA (p! independent from synthesis of mRNA Our study also 0.005) and GAPDH mRNA (P!0.003) levels were signifi- showed that 18S rRNA can be affected by trichostatin A (TSA) cantly reduced, however 18S rRNA and RPL13A mRNA levels at least in prostate and breast tissue and probably in other tissues as well, hence we would not recommend 18S rRNA as a In ZR-75-1 cells, at 100 ng/ml TSA, 18S rRNA (P!0.001), suitable housekeeping gene under the effects of histone b actin mRNA (p!0.001) and GAPDH mRNA (P!0.01) deacetylase inhibitors (HDACi) such as trichostatin A (TSA).
levels were significantly up-regulated in TSA treated samples, b actin mRNA remains a widely used housekeeping gene again RPL13A showed constant expression Simi- internal control in molecular biology despite the fact that larly, at 200 ng/ml TSA, 18S rRNA (P!0.05), and GAPDH many studies have reported its cell cycle dependent expression mRNA (P!0.04) levels were significantly altered in TSA pattern and regulation in specific circumstances. Some studies treated samples, however RPL13A mRNA and b actin mRNA have also questioned the use of b actin mRNA as suitable internal control in RT-PCR since it does not satisfy certain A. Mogal, S.A. Abdulkadir / Molecular and Cellular Probes 20 (2006) 81–86 Fig. 4. Quantitative RT-PCR analysis of housekeeping genes following trichostatin A (TSA) treatment in human breast cancer (T-47D and ZR-75-1) cell lines. (A)T-47D (100 ng/ml TSA): 18S rRNA (P!0.005), b actin mRNA (p!0.003) and GAPDH mRNA (P!0.01) levels were significantly reduced in TSA treated samplesas compared to the vehicle treated samples. However, RPL13A mRNA levels remained unaffected. (B) T-47D (200 ng/ml TSA): b actin mRNA (p!0.005) andGAPDH mRNA (P!0.003) levels were significantly reduced in TSA treated samples as compared to the vehicle treated samples. However 18S rRNA and RPL13AmRNA levels remained unaffected. (C) ZR-75-1 (100 ng/ml TSA): 18S rRNA (P!0.002), b actin mRNA (p!0.001) and GAPDH mRNA (P!0.01) levels weresignificantly up-regulated in TSA treated samples. On the other hand RPL13A mRNA remained unaffected and showed constant expression. (D) ZR-75-1 (200 ng/mlTSA): 18S rRNA (P!0.05), and GAPDH mRNA (P!0.04) levels were significantly altered in TSA treated samples, however RPL13A mRNA and b actin mRNAshowed constant expression. The data show the average of two different experiments performed in triplicate using the same conditions. (**P!0.005, *P!0.05).
basic requirements for application as a housekeeping gene prostate and breast tissue and probably in other tissues as well.
Further, some studies have specifically shown that using Thus, we would not recommend GAPDH mRNA as a suitable b actin mRNA as an internal control can detrimentally affect housekeeping gene with the use of trichostatin A (TSA).
the accuracy of RT-PCR results Similar to 18S rRNA, our Lastly we studied expression of the ribosomal highly basic study showed that b actin mRNA can also be regulated by 23-kDa protein (rb 23-kDa, RPL13A) following trichostatin A trichostatin A (TSA) at least in prostate and breast tissue and (TSA) treatment. Previous studies have shown that the probably in other tissues as well. Thus, we would not expression of the gene for the ribosomal highly basic 23-kDa recommend b actin mRNA as suitable housekeeping gene protein (RPL13A) was remarkably constant between different with the use of trichostatin A (TSA).
tissue types Furthermore, its expression was not affected Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a by malignant transformation or inflammation in the same tissue key enzyme in glycolysis, which makes it an abundant RNA in contrast to GAPDH One research group recommended species for use as a potential internal RNA standard. This RPL13A as a standard for normalization for at least the housekeeping gene is constitutively expressed in many tissues.
pancreas and prostate . In our study, the expression of this However, wide variation in GAPDH expression levels have gene was very stable following TSA treatment.
been observed in tissues at different developmental stages The stable and non-regulated expression of housekeeping in cells treated with insulin dexamethasone genes is critical for accurate interpretation of RT-PCR results.
mitogens as well as virally transformed or oncogene- Thus, it is essential to choose proper housekeeping genes when transfected fibroblasts Different tissue types exhibited normalizing RNA concentrations Our study supports the marked differences in the expression of GAPDH gene.
notion that no one housekeeping gene is perfect under all Furthermore, within the same tissue, GAPDH expression was experimental conditions, and that it is necessary to characterize up-regulated in the presence of inflammation or malignant the suitability of various housekeeping genes to serve as transformation . The up-regulated expression levels of internal controls under particular experimental conditions. In GAPDH have been previously reported in human pancreatic or conclusion, based on the results of our study, we recommend colon adenocarcinoma . In tumor cells this could be due to the use of the ribosomal highly basic 23-kDa protein (RPL13A) an increase in glycolysis and glucose turnover. Further, as a suitable standard for normalization with the use of histone GAPDH mRNA levels were up-regulated in the presence of deacetylase inhibitors (HDACi) such as trichostatin A (TSA).
hypoxia and by hypoxia inducible factor 1 (HIF-1) Moreover, GAPDH is pathologically implicated in neurode- generation and apoptosis . Our study showed that GAPDHmRNA can also be regulated by trichostatin A (TSA) at least in Supported by grant CA94858 from the NCI (SAA).
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Reverse shoulder arthroplasty combined with latissimus dorsi transfer using the bone-chip technique. Ortmaier R, Resch H, Hitzl W, Mayer M, Blocher M, Vasvary I, Mattiassich G, Stundner O, Tauber M . Int Orthop. 2013 Oct 17. [Epub ahead of print] PMID: 24132802 [PubMed - as supplied by publisher] Related citations Select item 24075998 2. Clinical and computed tomography-radiologic outc