Abstract
Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA.
Highlights
Induction of urokinase plasminogen ogen activator (uPA) expression by histone deacetylase (HDAC) Tumor invasion is mediated by uPA through the conversion inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid of plasminogen to plasmin, which degrades basement memwas observed in all three different types of human cancer cells branes [11, 12]
Mounting activator gene expression are not fully understood. In this evidence from laboratories suggests a role for uPA in the invastudy, we investigated whether histone deacetylation was sion of cancer cells as well as the risk for a relapse in cancer involved in repression of uPA expression in human cancer cells. patients (6 –10)
Induction of uPA expression by histone deacetylase (HDAC) Tumor invasion is mediated by uPA through the conversion inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid of plasminogen to plasmin, which degrades basement memwas observed in all three different types of human cancer cells branes [11, 12]
Summary
Reagents—TSA, SCR, and 5-aza-2Ј-deoxycytidine (5-aza) were purchased from Sigma. TSA and SCR were dissolved in Me2SO; 5-aza was dissolved in phosphate-buffered saline. Fibrin Zymography—The enzymatic activity and molecular weight of electrophoretically separated forms of uPA were determined in conditioned medium of human cancer cell lines SK-N-BE, SK-N-AS, SF-3061, and LNCaP by SDS-PAGE as described previously [30]. The cells were washed twice with ice-cold phosphatebuffered saline containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, and 1 g/ml pepstatin A), harvested, and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths below 1000 bp (amplitude 60%, 4 ϫ 10 s, Fisher Sonic Dismembrator 60, Pittsburgh, PA). DNA from PvuII-digested nuclei was amplified by PCR with primers uPA-F1 (5ЈCAG GTG CAT GGG AGG AAG CA-3Ј) and uPA-R (5Ј-GGC CAC CGG GAC TGC CCC AG-3Ј) and electrophoresed on a 2% agarose gel (Fig. 3A). Cells were detached, washed twice in phosphate-buffered saline, and resuspended in serum-free advanced Dulbecco’s modified Eagle’s medium. Values are expressed as mean Ϯ S.D. from at least three separate experiments, and differences were considered significant at a p value of Ͻ0.05
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