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-Class Glutathione S-Transferase (GSTP1) Expression in Human Prostate Cancer Cells by Treatment with Procainamide1
Departments of Oncology [X. L., K. A., W. R. G., X. Y., B. S. C., A. K., S. P., T. L. D., A. M. D., W. G. N.], Pathology [M. J. P., W. R. G., A. M. D., W. G. N.], Urology [T. L. D., A. M. D., W. G. N.], Pharmacology [W. G. N.], and Medicine [W. G. N.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
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ABSTRACT |
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-class glutathione S-transferase (GSTP1), and fail to express GSTP1 as a consequence of transcriptional "silencing." Inactivation of GSTP1 by CpG island hypermethylation, the most common somatic genome alteration yet reported for human PCAs, occurs early during human prostatic carcinogenesis and results in a loss of GSTP1 "caretaker" function, leaving prostate cells with inadequate defenses against oxidant and electrophile carcinogens. We report here that the drug procainamide, a nonnucleoside inhibitor of DNA methyltransferases, reversed GSTP1 CpG island hypermethylation and restored GSTP1 expression in LNCaP human PCA cells propagated in vitro or in vivo as xenograft tumors in athymic nude mice. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Nucleoside analogue inhibitors of DNA methyltransferases, such as 5-aza-C3 and 5-aza-dC, have been widely used in attempts to reverse abnormal DNA methylation changes in cancer cells and restore "silenced" gene expression (5 , 6) . Unfortunately, despite some apparent successes using preclinical models and some promising results in early clinical trials, the clinical utility of these compounds has not yet been fully realized (6) . One of the limitations of the nucleoside analogue methyltransferase inhibitors in clinical trials has been treatment-associated side effects, such as myelotoxicity with resultant neutropenia and thrombocytopenia, which are characteristic of other nucleoside analogues in general, including nucleoside analogues that are not DNA methyltransferase inhibitors (6) . Another concern about the use of nucleoside analogues as DNA methyltransferase inhibitors has been that incorporation of the nucleoside analogues into genomic DNA might lead to mutations and/or cancer development (7, 8, 9, 10, 11, 12) . This has prompted efforts at discovery and development of nonnucleoside analogue DNA methyltransferase inhibitors, such as DNMT1 antisense preparations and other agents, which might attenuate DNA methyltransferase activity with less treatment-associated toxicity (13 , 14) . We report here that the drug procainamide, a nonnucleoside inhibitor of DNA methyltransferases (15) approved by the United States Food and Drug Administration for the treatment of cardiac arrhythmias, reversed GSTP1 CpG island hypermethylation, the most common somatic genome change in human prostate cancer (PCA; Refs. 16, 17, 18 ), and restored GSTP1 expression in LNCaP human PCA cells propagated in vitro or in vivo as xenograft tumors in athymic nude mice.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Detection of GSTP1 mRNA Using Quantitative RT-PCR.
Total RNA was isolated from LNCaP PCA cells using an Rneasy RNA isolation kit (Qiagen). GSTP1 mRNA was detected by quantitative RT-PCR using an iCycler iQ multi-color real time PCR system (Bio-Rad). Before PCR, cDNA was synthesized from 1 µg of RNA using an Omniscript RT kit (Qiagen). PCR reaction mixtures included cDNA from 125 ng of RNA, sense (5'-GGGCAGTGCCTTCACATAGT-3') and antisense (5'-GGAGACCTCACCCTGTACCA-3') primers, and the Master Mix from a QuantiTect SYBR Green PCR kit (Qiagen). PCR cycles involved incubation at 94°C for 30 s, at 60°C for 30 s, and then at 72°C for 30 s. Cloned GSTP1 cDNA was used as a standard for quantification. As a control, TBP mRNA, encoding TATA binding protein, was also detected by quantitative RT-PCR, using specific sense (5'-CACGAACCACGGCACTGATT-3') and antisense (5'-TTTTCTTGCTGCCAGTCTGGAC-3') primers and the same PCR reaction mixture (21)
. PCR cycles for TBP cDNA detection involved incubation at 94°C for 30 s, at 55°C for 30 s, and then at 72°C for 30 s. Cloned TBP cDNA was used as a standard for quantification. Each of the PCR assays was run in triplicate; GSTP1 and TBP mRNA copy numbers were estimated from the threshold amplification cycle numbers using software supplied with the iCycler iQ Thermal Cycler.
Immunohistochemical Detection of GSTP1 Polypeptides in LNCaP PCA Xenograft Tissue Sections.
Formalin-fixed, paraffin-embedded LNCaP PCA xenograft tumor tissues were cut into 5-µm sections and stained with anti-GSTP1 antibodies (1:3000 dilution; Dako), using an immunoperoxidase method (ChemMate Universal Detection System; Ventana Medical Systems) with diaminobenzidine as a peroxidase substrate (18
, 22) . Immunostained tissue sections were counterstained with hematoxylin.
MSP for the Detection of Hypermethylated and of Unmethylated GSTP1 CpG Island Alleles.
Genomic DNA was isolated from LNCaP PCA cells using a QIAamp DNA isolation kit (Qiagen). Purified DNA (25 ng) was treated with XhoI and XbaI, admixed with salmon sperm DNA (2.5 µg), and then exposed to sodium bisulfite to permit MSP as described previously (23)
. Bisulfite-treated DNA was then subjected to MSP using primers selective for unmethylated (5'-GATGTTTGGGGTGTAGTGGTTGTT-3' and 5'-CCACCCCAATACTAAATCACAACA-3') and for methylated (5'-TTCGGGGTGTAGCGGTCGT-3' and 5'-GCCCCAATACTAAATCACGACG-3') GSTP1 CpG island sequences (23)
. PCR reaction mixtures contained the AmpliTaqGold polymerase in GeneAmp reaction buffer (Applied Biosystems), 2.5 mM MgCl2, 250 µM deoxynucleotide triphosphates, and 1 µM primers. PCR cycles involved incubation at 94°C for 15 s, at 62°C for 30 s, and then at 72°C for 30 s; 45 PCR cycles were undertaken. PCR reaction products were electrophoresed on agarose gels and visualized by ethidium bromide staining.
Bisulfite Genomic Sequencing for the Assessment of Somatic GSTP1 CpG Island DNA Methylation in LNCaP PCA Xenograft Tumors.
Genomic DNA was isolated from the LNCaP xenograft tumors using the DNeasy DNA isolation kit (Qiagen). To map CpG dinucleotide changes throughout the GSTP1 "CpG island," bisulfite genomic sequencing, which permits discrimination of 5-mC from C, was undertaken (24)
. Purified DNAs (200 ng) were treated with EcoRI, admixed with salmon sperm DNA (2.5 µg), and then exposed to sodium bisulfite. Bisulfite-treated DNA was then subjected to two rounds of PCR to amplify GSTP1 CpG island alleles, using primers that recognize "antisense" strand GSTP1 sequences after conversion of C to T (first PCR reaction primers: GenBank positions -636 to -613, 5'-ACA/GCAACCTATAATTCCACCTACTC-3', and +117 to +94, 5'-GTT/CGGGAGTTGGGGTTTGATGTTG-3'; second PCR reaction primers: GenBank positions -535 to -512, 5'-AACCTAAACCACAACA/GTAAAACAT-3', and +89 to +66, 5'-TTGGTTTTATGTTGGGAGTTTTGA-3') and using reaction conditions that have been described previously. To permit DNA sequencing of individual GSTP1 CpG island alleles, the PCR products were purified by electrophoresis on 1% agarose gels (Life Technologies, Inc.), isolated from the agarose (using a QIAquick gel extraction kit; Qiagen), recovered by ethanol precipitation, and then cloned by ligation into pCR 2.1pTOPO cloning vectors (using a TOPO kit; Invitrogen), followed by introduction into TOP 10 One Shot competent bacteria. Plasmid DNAs isolated from independent drug-resistant bacterial clones (a minimum of four clones for each PCR reaction product) were subjected to DNA sequence analysis using a cycle sequencing approach with M13 sequencing primers dye-labeled terminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit; Perkin-Elmer) and an ABI automated sequencer.
Effects of Procainamide and N-Acetyl-procainamide on GSTP1 Promoter Function, on Histone Acetylation, and on Gene Expression in LNCaP PCA Cells.
To ascertain whether procainamide and N-acetyl-procainamide exposure might trigger trans-activation of the GSTP1 promoter in LNCaP PCA cells or other human cancer cells, LNCaP PCA cells, PC-3 PCA cells (25)
, MCF-7 breast cancer (BCA) cells (26)
, and HCT116 colorectal cancer (CRC) cells (27)
were transfected with pCMV ß-Gal (Promega), with pGL3-Control (containing SV40 promoter sequences; Promega), or with pGL-GSTP1 (prepared by cloning GSTP1 promoter sequences, GenBank positions -408 to +36, into pGL3-Basic; Promega) in a manner described previously. The transfected cells were then treated for 24 h with and without procainamide and N-acetyl-procainamide in complete growth medium. To assess reporter gene expression, the transfected cells were lysed and then assayed for ß-galactosidase activity (using the ß-Galactosidase Enzyme Assay System with Reporter Lysis Buffer; Promega) or for luciferase activity (using the Luciferase Assay System with Reporter Lysis Buffer; Promega). To determine whether procainamide or N-acetyl-procainamide causes changes in histone acetylation, histone-containing protein extracts from LNCaP PCA cells that had been treated with the agents, or treated with the histone deacetylase inhibitor trichostatin A, were subjected to immunoblot analysis for histone acetylation, using rabbit antibodies specific for total H4 histone, for acetylated H4 histone, for acetyl-lysine 5 H4 histone, for acetyl-lysine 8 H4 histone, and for acetyl-lysine 12 H4 histone (Serotec, Inc.). Total protein extracts, prepared by lysing the cells in buffer containing 2% SDS, were separated on a 10% polyacrylamide gel with 2-(N-morpholino)ethanesulfonic acid running buffer and then transferred to nitrocellulose membranes (NOVEX; Invitrogen). The membranes were then probed with each of the rabbit anti-H4 histone antibodies, using antibody concentrations recommended by the manufacturer (Serotec, Inc.). Rabbit anti-H4 histone antibody binding was detected using horseradish peroxidase-conjugated IgG (Amersham) and SuperSignal West Pico Chemiluminescent Substrate (Pierce).
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Both procainamide and N-acetyl-procainamide, a major procainamide metabolite, have been shown to inhibit DNA methyltransferase activity in extracts from mammalian cells (15)
. In addition, although the mechanism by which each of the agents effect enzyme inhibition has not been established, both clearly act differently than 5-aza-C and 5-aza-dC, appearing to bind to GC-rich DNA rather than to be incorporated into the DNA template (28
, 29)
. To ascertain whether procainamide could reverse GSTP1 CpG island hypermethylation and restore GSTP1 expression in LNCaP PCA cells, growing cultures of LNCaP PCA cells were treated with procainamide at a concentration of 100 µM, treated with N-acetyl-procainamide at a concentration of 100 µM, or treated with 5-aza-dC at a concentration of 10 µM, continuously for 2 weeks. Quantitative RT-PCR revealed that exposure to procainamide or to 5-aza-dC, but not to N-acetyl-procainamide, resulted in the appearance of GSTP1 mRNA in LNCaP PCA cells (Fig. 1A)
. Of interest, although GSTP1 expression was evident after 1 week of procainamide treatment, GSTP1 mRNA did not appear until after 2 weeks of exposure to 5-aza-dC treatment. Restoration of GSTP1 expression in LNCaP PCA cells treated with procainamide or with 5-aza-dC was accompanied by the appearance of unmethylated GSTP1 CpG island alleles in LNCaP PCA cell DNA, as detected using a MSP technique (Fig. 1B
; Ref. 23
).
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. In fact, procainamide appeared as effective as 5-aza-dC at restoring GSTP1 expression at the doses and dosing schedule used (Fig. 2D)
. The restoration of GSTP1 expression in LNCaP PCA cells by procainamide treatment appeared likely to be a consequence of DNA methyltransferase inhibition, because GSTP1 reactivation was accompanied by a reduction in GSTP1 CpG island hypermethylation detected by bisulfite genomic sequencing analysis (Fig. 3)
. Of interest, even in the absence of treatment with procainamide or with 5-aza-dC, propagation of LNCaP PCA cells in vivo as xenograft tumors in immunodeficient mice resulted in a slight reduction of GSTP1 CpG island hypermethylation (Fig. 3)
and in the reactivation of GSTP1 expression in a small number of cells (Fig. 2, A and D)
. The mechanism for this phenomenon, not seen when LNCaP PCA cells are propagated in vitro, was not determined. In a previous study, LNCaP PCA cells and LNCaP cells genetically modified to express high GSTP1 levels by transfection of GSTP1 cDNA exhibited similar growth properties in vitro and in vivo. Perhaps not surprisingly then, neither procainamide nor 5-aza-dC had any dramatic effects on LNCaP PCA xenograft tumor growth properties in vivo (Fig. 2E)
. Nonetheless, procainamide appeared slightly more effective than 5-aza-dC at limiting LNCaP PCA xenograft tumor growth, although procainamide was not more effective than 5-aza-dC at reactivating GSTP1 expression (Fig. 2, D and E)
. Whether the mild inhibition of LNCaP PCA xenograft tumor growth by procainamide was a consequence of DNA methyltransferase inhibition, such as via the reactivation of a "silenced" gene (or genes) other than GSTP1 involved in growth control, or was the result of some other action of the drug, was not discerned.
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. The induction of GSTP1 promoter activity by trichostatin A was far greater (
60 fold) than the induction by procainamide (<2 fold) or by N-acetyl-procainamide (>3 fold). The effect of N-acetyl-procainamide treatment on GSTP1 promoter function was not specific; N-acetyl-procainamide triggered increases in the activities of CMV and SV40 promoters in LNCaP PCA cells (Fig. 4B)
and augmented CMV promoter activity in PC-3 PCA cells, in MCF-7 BCA cells, and in HCT116 colorectal cancer (CRC) cells (Fig. 4C)
. To determine whether either procainamide or N-acetyl-procainamide might affect histone acetylation or deacetylation, LNCaP PCA cells were treated with procainamide, with N-acetyl-procainamide, or with the histone deacetylase inhibitor trichostatin A and then subjected to immunoblot analysis for acetylated histones (Fig. 5)
. As expected, trichostatin A treatment increased histone H4 acetylation, particularly at lysine 5 and at lysine 8 (Fig. 5)
. In contrast, exposure to procainamide or to N-acetyl-procainamide failed to have much of an effect on overall histone H4 acetylation or on acetylation at lysine 5 or at lysine 8 (Fig. 5)
. Curiously, procainamide treatment, and not treatment with either trichostatin A nor with N-acetyl-procainamide, appeared to slightly increase H4 acetylation at lysine 12 (Fig. 5)
. Considering all of the data collected, the only evidence that procainamide or N-acetyl-procainamide might activate GSTP1 expression in LNCaP PCA cells in vivo by any mechanism other than DNA methyltransferase inhibition was that the agents were able to increase GSTP1 promoter activity slightly in LNCaP PCA cells when provided at a high concentration in vitro (Fig. 4)
. However, it is unlikely that either agent was able to increase GSTP1 promoter activity in LNCaP PCA cells in vivo by the same mechanism. The N-acetyl-procainamide augmentation of CMV promoter activity in vitro occurred at N-acetyl-procainamide concentrations (>100 µM; see Fig. 4D
) higher than that usually achieved in vivo accompanying procainamide treatment (4–8 µg/ml or 14.7 to 29.4 µM). Thus, the reactivation of GSTP1 in LNCaP PCA xenograft tumors in vivo accompanying procainamide treatment was most likely attributable to drug inhibition of DNA methyltransferases and not to drug effects on histone acetylation or other transcriptional regulatory process.
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Somatic CpG island DNA methylation changes have been reported to occur very early during the pathogenesis of many human cancers, suggesting that DNA methyltransferase inhibitors might be considered for use as cancer chemoprevention drugs as well as cancer treatment drugs, if inhibitors with adequate safety profiles can be discovered (4 , 41 , 42) . In support of this concept, 5-aza-dC treatment reduced the appearance of intestinal polyps, thought to represent premalignant lesions, in ApcMin/+ mice (4) . The recognition in this report that both 5-aza-dC and procainamide may be capable of "silenced" gene reactivation in cancer cells, despite exhibiting markedly different side effects, suggests that relatively nontoxic DNA methyltransferase inhibitors can likely be developed for use in cancer prevention as well as cancer treatment. The one side effect possibly shared by 5-aza-dC and procainamide may be the induction of autoimmunity (43) . However, whether autoimmunity constitutes an unavoidable side effect of DNA methyltransferase inhibition is not known. Finally, although procainamide has been a fairly widely used drug for many years, no data have been published regarding the effects of the drug on cancer development. Perhaps, an epidemiology study of cancer incidence and mortality associated with procainamide treatment might disclose whether DNA methyltransferase inhibitor treatment reduces or increases cancer risks.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by an Award from the Association for Cure of Cancer of the Prostate (CaP CURE) and by NIH/NCI Grants CA58236 and CA70196. 
2 To whom requests for reprints should be addressed, at Bunting-Blaustein Cancer Research Building, Room 151, 1650 Orleans Street, Baltimore, MD 21231-1000. Phone: (410) 614-1661; Fax: (410) 502-9817; E-mail: bnelson{at}jhmi.edu
3 The abbreviations used are: 5-aza-C, 5-aza-cytidine; 5-aza-dC, 5-aza-deoxycytidine; GSTP1, -class glutathione S-transferase; RT-PCR, reverse transcription-PCR; MSP, methylation-specific PCR; CMV, cytomegalovirus.
4 X. Lin et al., unpublished results.
Received 4/26/01. Accepted 10/30/01.
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) or after 2 weeks (14-day treatment; 
) of exposure was subjected to analysis for GSTP1 mRNA expression by quantitative RT-PCR (see "Materials and Methods"). Data shown are GSTP1 mRNA copy number/TBP mRNA copy number; bars, ± SE. In addition, for each treatment condition in B, genomic DNA isolated from LNCaP PCA cells after 2 weeks of treatment was subjected to analysis for GSTP1 CpG island methylation using a MSP assay (see "Materials and Methods"). Displayed are PCR products generated with primers specific for unmethylated GSTP1 CpG island alleles (U) and for hypermethylated GSTP1 CpG island alleles (M).
; plotted using SigmaPlot 5.0 software). A significant (P < 0.0001) increase in the fraction of LNCaP PCA cells expressing GSTP1 was observed after treatment either with procainamide or with 5-aza-dC. In E, the mean size (bars, SE) of LNCaP PCA xenograft tumors after treatment with PBS, procainamide, and 5-aza-dC are shown. A significant reduction in tumor size was seen after treatment with procainamide (P < 0.006) but not after treatment with 5-aza-dC (P = 0.275).
, CpG.
