Lycopene and apo-10'-lycopenal do not alter DNA methylation of GSTP1 in LNCaP cells

DNA hypermethylation and silencing of tumor-suppressor genes are commonly seen in human cancers, and likely contribute to the process of carcinogenesis. A growing body of evidence suggests that dietary compounds may alter cancer risk through epigenetic modifications. Glutathione S-transferase P1 (GSTP1) is hypermethylated in >90% of prostate cancer cases making it one of the most common genome alterations in prostate cancer. LNCaP cells were treated either with lycopene or apo-10′-lycopenal for 7 days, and mRNA expression and DNA methylation of GSTP1 were evaluated. Neither compound significantly altered expression nor methylation of GSTP1 while treatment with 5-azacytidine decreased methylation by 78%. These findings demonstrate that lycopene and apo-10′-lycopenal are not effective demethylating agents of GSTP1 in the human LNCaP cell line.     

Demethylating agent 5-aza-2-deoxycytidine enhances susceptibility of breast cancer cells to anticancer agents

DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko’s fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 μM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARβ2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.     

DNA hypermethylation as a chemotherapy target

Epigenetics refers to partially reversible, somatically inheritable, but DNA sequence-independent traits that modulate gene expression, chromatin structure, and cell functions such as cell cycle and apoptosis. DNA methylation is an example of a crucial epigenetic event; aberrant DNA methylation patterns are frequently found in human malignancies. DNA hypermethylation and the associated expression silencing of tumor suppressor genes represent a hallmark of neoplastic cells. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. Several small synthetic and natural molecules, are able to reverse the DNA hypermethylation through inhibition of DNA methyltransferase (DNMT). DNMT is the enzyme catalyzing the transfer of methyl groups to cytosines in genomic DNA. These reagents are studied intensively in cell cultures, animal models, and clinical trials for potential anti-cancer activities. It was found that accompanying DNA demethylation is a dramatic reactivation of the silenced genes and inhibition of cancer cell proliferation, promotion of cell apoptosis, or sensitization of cells to other chemotherapeutic reagents. During the last few decades, an increasing number of DNMT inhibitors (DNMTi) targeting DNA methylation have been developed to increase efficacy with reduced toxicity. This review provides an update on new findings on cancer epigenetic mechanisms, the development of new DNMTi, and their application in the clinical setting. Current challenges, potential solutions, and future directions concerning the development of DNMTi are also discussed in this review.     

Insensitivity to transforming growth factor-beta results from promoter methylation of cognate receptors in human prostate cancer cells (LNCaP)

Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.     

Lycopene inhibits the proliferation of androgen-dependent human prostate tumor cells through activation of PPARγ-LXRα-ABCA1 pathway

The activation of nuclear receptors, peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα), has been shown to inhibit the growth of prostate cancer cells. This study examined whether the anti-proliferative effect of lycopene on androgen-dependent human prostate cancer (LNCaP) cells involves the up-regulation of the expression of PPARγ and LXRα. As expected, lycopene treatment (2.5-10 μM) significantly inhibited the proliferation of LNCaP cells during incubation for 96 h. Lycopene significantly increased the protein and mRNA expression of PPARγ and LXRα at 24 and 48 h, while the increased in the expression of ATP-binding cassette transporter 1 (ABCA1) was only evident 96 h. In addition, lycopene significantly decreased cellular total cholesterol levels and increased apoA1 protein expression at 96 h. Incubation of LNCaP cells with lycopene (10 μM) in the presence (20 μM) of a specific antagonist of PPARγ (GW9662) and LXRα (GGPP) restored the proliferation of LNCaP cells to the control levels and significantly suppressed protein expression of PPARγ and LXRα as well as increased cellular total cholesterol levels. LXRα knockdown by siRNA against LXRα significantly enhanced the proliferation of LNCaP cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. The present study is the first to demonstrate that the anti-proliferative effect of lycopene on LNCaP cells involves the activation of the PPARγ-LXRα-ABCA1 pathway, leading to reduced cellular total cholesterol levels.     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.     

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16^INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.     

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.     

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background

The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and Principal Findings

Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions

The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Different effect of beta-carotene on proliferation of prostate cancer cells

It was shown that high doses of beta-carotene (>30 microM) decrease proliferation of prostate cancer cells in vitro. However, it is rather doubtful whether such concentration of beta-carotene is really accessible at cellular level. We studied the effect of 3 and 10 microM beta-carotene on proliferation and gene expression in LNCaP and PC-3 prostate cancer cell lines. Beta-carotene--more efficiently absorbed from medium by androgen-sensitive LNCaP cells--increased proliferation of LNCaP cells whereas it had weaker effect on PC-3 cells. Initial global analysis of expression of genes in both cell lines treated with 10 microM beta-carotene (Affymetrix HG-U133A) showed remarkable differences in number of responsive genes. Their recognition allows for conclusion that differences between prostate cancer cell lines in response to beta-carotene treatment are due to various androgen sensitivities of LNCaP and PC-3 cells. Detailed analysis of expression of selected genes in beta-carotene treated LNCaP cells at the level of mRNA and protein indicated that the observed increase of proliferation could have been the result of slight induction of a few genes affecting proliferation (c-myc, c-jun) and apoptosis (bcl-2) with no significant effect on major cell cycle control genes (cdk2, RB, E2F-1).     

Differential expression of galectins in normal, benign and malignant prostate epithelial cells: silencing of galectin-3 expression in prostate cancer by its promoter methylation

Galectins (gal), a family of soluble beta-galactoside-binding proteins present at the cell surface, are involved in cancer progression and metastasis. Here we investigated the expression of several galectins in normal (PrEC), benign (BPH-1), and malignant (LNCaP) prostate epithelial cells and found that all galectins, except gal1 are differentially expressed. The gal3, 7, and 9 are highly expressed in PrEC, but not in LNCaP cells. Out of seven isoforms of gal8, the proto isoform gal8e and our newly discovered proto isoform gal8g were upregulated in LNCaP cells compared to PrEC, whereas the two tandem-repeat isoforms gal8a and gal8b were equally expressed in these cells. To determine if the silencing of gal3 in LNCaP cells was due to promoter methylation, LNCaP cells were treated with azacytidine. Azacytidine treatment induced the expression of gal3 in LNCaP cells, indicating that the gal3 gene was silenced by methylation of its promoter. To examine further, we evaluated cytosine methylation in gal3 promoter in LNCaP, normal prostate and placenta DNA and observed that it is highly methylated in LNCaP but not in normal cells and azacytidine completely abolished this methylation in LNCaP cells. Similar to prostate cancer cells, gal3 promoter was highly methylated in human prostate cancer tissue but not in normal tissue. To our knowledge, this is the first report indicating that gal3 expression is regulated by promoter methylation in LNCaP cells and prostate tumors. The methylation of gal3 promoter may constitute a powerful tool for early diagnosis of prostate cancer.     

Involvement of Cdk5/p25 in digoxin-triggered prostate cancer cell apoptosis

Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca(2+) proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca(2+), and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.     

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells

5-Aza-2'-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [(3)H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as gamma-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.     

MethylScreen: DNA methylation density monitoring using quantitative PCR

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.