Molecular Cancer Research 5, 873-880, September 1, 2007. Published Online First August 17, 2007;
doi: 10.1158/1541-7786.MCR-06-0141
© 2007 American Association for Cancer Research
Angiogenesis, Metastasis, and the Cellular Microenvironment
Down-Regulation of Placenta Growth Factor by Promoter Hypermethylation in Human Lung and Colon Carcinoma
Lei Xu and
Rakesh K. Jain
Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts
Requests for reprints: Lei Xu, Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, 100 Blossom Street, Cox-7, Boston, MA 02114. Phone: 617-726-8051; Fax: 617-726-1962. E-mail: lei{at}steele.mgh.harvard.edu
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Abstract
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Two recent clinical trials have shown that the placenta growth factor (PlGF) is up-regulated after bevacizumab treatment in colorectal cancer and after SU11248 treatment in metastatic renal cell carcinoma. The regulation of expression for the vascular endothelial growth factor (VEGF) has been well documented in human tumors; however, the data for PlGF are lacking. We investigated the epigenetic regulation of PlGF and correlated the results with clinicopathologic features. We used plgf promoter analysis, cDNA microarray, immunohistochemistry, and Northern blot analysis to determine the expression level of PlGF in 22 human lung carcinoma and 11 colorectal tumors and in 12 cell lines. Sodium bisulfite modification of genomic DNA followed by methylation-specific PCR (MSP) and sequencing were used to determine the methylation status of the PlGF promoter. Treatments with 5-aza-2'-deoxycytidine and trichostatin A (TSA) were used to reactivate PlGF expression. Significance analysis showed that PlGF expression level was low in human lung and colorectal tumor tissues and in cell lines. PlGF gene promoter was hypermethylated. Treatment with the demethylating agent 5-Aza-dC restored PlGF transcript expression in the lung and colon carcinoma cell lines. By combining the results from cDNA microarray, immunohistochemistry, and MSP, we report, for the first time, that the PlGF gene promoter is methylated, and methylation may be one of the mechanisms that contributes to the low PlGF expression level in human lung and colorectal tumor tissues and cell lines. (Mol Cancer Res 2007;5(9):873–80)
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Introduction
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The vascular endothelial growth factor (VEGF) family consists of seven structurally related members, including VEGF-A, PlGF, VEGF-B, VEGF-C and VEGF-D, VEGF-E, and snake venom VEGF (1). VEGF-A was first characterized as a mitogen for endothelial cells and inducing vessel permeability (2). It is well documented that VEGF is essential for normal vascular development and promotes angiogenesis in a variety of in vitro and in vivo settings by modulating proliferation, permeability, and survival (3). Placenta growth factor (PlGF) was discovered 2 years after the cloning of VEGF (4). The role of PlGF in pathologic angiogenesis is controversial. Although some studies showed that PlGF enhances pathologic angiogenesis by initiating crosstalk between VEGFR-1 and VEGFR-2 (5-7), others showed that PlGF inhibits tumor growth and angiogenesis (8, 9). Our recent studies showed that overexpression of PlGF inhibits tumor growth, angiogenesis, and metastasis in human lung and colon orthotopical tumor models (10).
VEGF is expressed in a variety of cell types, whereas the expression of PlGF is limited to the placental tissue (11). In different tumors, VEGF have been shown to be significantly up-regulated in breast, ovarian, colon, kidney, and bladder carcinomas as well as in some brain tumors (12). In human thyroid carcinoma cell lines, VEGF expression is elevated in cells of different tumorigenic potential, whereas PlGF expression is down-regulated (13). In hypervascular germ cell tumors and hemangioblastoma tumors, VEGF is dramatically up-regulated in the majority of tumors, whereas PlGF level is generally very low and is only detected in a small number of cases (14-17). Several recent studies showed PlGF expression correlates with clinical prognosis in non–small cell lung cancer and in colorectal cancer (18, 19). Furthermore, two recent clinical trials showed that PlGF levels are elevated in the plasma of colorectal and rectal carcinoma patients receiving bevacizumab or SU11248 (20, 21). However, very little is known about the regulation and the molecular mechanisms of PlGF.
Epigenetic mechanisms have been shown to silence tumor suppressor genes in a variety of human cancers (22). Several studies have shown that various genes are hypermethylated and silenced in non–small cell lung cancer and colorectal cancer. These include genes involved in cell cycle regulation and angiogenesis (cyclooxygenase-2, p14, and p16), DNA repair or protection (hMLH1, MGMT, and GSTP1), signal transduction (APC and RASS-F1A), and those related to metastasis and invasion [E-cadherin and tissue inhibitor of metalloproteinase (TIMP)-3; refs. 23, 24]. However, the mechanisms that contribute to PlGF expression regulation are not known.
Here, we examined PlGF expression and its regulation in human lung and colon carcinoma. Our study showed that PlGF expression was low in human lung and colon carcinoma cell lines and tumor tissues, and that the low expression was associated with aberrant 5' CpG methylation of the PlGF promoter. PlGF promoter methylation was detected in 22 of the lung and 11 colorectal carcinoma tumors analyzed. Treatment with the demethylating agent 5-Aza-dC restored PlGF transcription in the lung and colon carcinoma cell lines, confirming that the low level expression of this gene in the cell lines was a result of methylation.
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Results
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Microarray Analysis Identifies that PlGF Expression Is Low in Human Lung and Colon Carcinomas
To screen the PlGF expression level in human clinical tumors, a specific Plgf probe was radioactively labeled and hybridized onto the disease-profiling array II containing 154 cDNA pair samples derived from multiple human tumors and corresponding normal tissue from individual patients. Signal intensities were calculated for individual spots using a FluoChem 8800 (Alpha Innotech). This array revealed that hybridization with PlGF probe gave a weak signal in most of the tumor types, including breast, ovary, stomach, prostate, bladder, uterus, cervix, rectum, thyroid gland, testis, skin, small intestine, trachea, and liver cancer (data not shown). Most significantly, PlGF signal was weaker in all of the 10 lung carcinomas and all of the 10 colon carcinoma samples when compared with the corresponding normal tissue cDNA. In contrast, a significant up-regulation of VEGF was observed in all of the lung and colon tumors compared with the corresponding normal tissue cDNA (Fig. 1A
).
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FIGURE 1. Expression of PlGF and VEGF in human lung and colon carcinoma cDNA and tumor cells. A. Disease profiling array analysis of PlGF and VEGF expression in 10 human colon carcinoma and 10 lung carcinoma cDNA. N = normal; T = tumor. Numbers shown are densitometry data, which showed the relative level of tumor PlGF level as compared with that of the corresponding normal tissue (defined as 1). B. Tumor cells were cultured under confluent conditions. mRNA was extracted, and a Northern blot analysis was done. Equal loadings were determined by normalization to ß-actin. C. Supernatant from confluent tumor cell cultures were collected and analyzed by ELISA. Columns, mean representative of at least three independent experiments; bars, SE.
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PlGF Expression Is Low in Human Lung and Colon Carcinoma Cell Lines
We screened the expression level of PlGF in human lung and colon carcinoma cell lines. Six cell lines of each tumor type were cultured and lysed for extraction of mRNA and protein. BeWo, a trophoblast cell line, which has been reported to express high levels of PlGF, was used as positive control. Low expression levels of PlGF mRNA expression in 12 tumor cell lines were noted by Northern blot analysis (Fig. 1B). To investigate whether these cells express and secrete PlGF protein, conditioned medium was collected from 12 cell lines, and PlGF and VEGF protein levels were measured by ELISA. All of the cell lines examined express high levels of VEGF protein, whereas PlGF protein expression was low (Fig. 1C).
PlGF Expression Is Low in Human Lung and Colon Carcinoma Tumor Tissues
To investigate whether PlGF expression was also low in human lung and colon tumors, histologic sections from 22 lung carcinoma and 11 colorectal adenocarcinoma clinical samples were stained with an antibody against human PlGF (Tables 1
and 2
). In normal lung tissues, PlGF is highly expressed in the epithelium of the intrapulmonary bronchus and alveolar and in the alveolar macrophages (Fig. 2A
). They displayed strong and diffuse cytoplasmic staining. PlGF is expressed at very low levels in the lung carcinoma tumor tissues (Fig. 2B). In normal colorectal tissues, PlGF is highly expressed in the cytoplasm of the epithelium (Fig. 2C). Under the same staining conditions, in poorly differentiated adenocarcinoma (Fig. 2D), PlGF expression was very low and not detected in most of the tumor tissues. In medium- to well-differentiated adenocarcinomas with a clearly defined glandular pattern, PlGF is expressed in the epithelium as well (Fig. 2E).
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FIGURE 2. Immunohistochemical analysis of PlGF expression in human tissues. A. In normal human lung tissue, (B) in lung carcinoma, (C) in normal colorectal tissue, (D) in poorly differentiated colorectal carcinoma, (E) in moderately differentiated colorectal carcinoma. A, Alveoli; M, alveolar macrophages; T, terminal bronchiole; Tu, tumor tissue. Bar, 100 µm.
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PlGF Promoter Activity in Lung and Colon Carcinoma Cell Lines
To study if the reduced PlGF mRNA expression was due to the loss of PlGF promoter activity, 3.2 kb of the PlGF promoter region was subcloned and tested using the luciferase reporter gene. Lung carcinoma cell lines, A549, NCI-H1299, and colon carcinoma cell lines, HCT116 and LS174T were transiently transfected with pGL3-basic or with pGL3-plgf. Figure 3
shows that PlGF promoter activities were 78-, 94-, 104- and 88-fold higher than the promoterless luciferase reporter (pGL3-basic) in A549, NCI-H1299, HCT116, and LS174T, respectively. This result suggests that transcription factors necessary for PlGF expression are present and able to support PlGF promoter activity in lung and colon carcinoma cells.
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FIGURE 3. PlGF promoter activity analysis by luciferase reporter gene assay. A549, NCI-H1299, HCT116, and LS174T cells were transiently transfected with PlGF-luciferase or pGL3-Basic luciferase reporter gene. The normalized luciferase activities are the luminometer readings of firefly luciferase activity normalized for transfection efficiency on the basis of Renilla luciferase activity. *, statistically significant difference (P < 0.001). Columns, mean of three independent experiments; bars, SD.
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Methylation Status of Plgf Promoter in Lung and Colon Carcinoma Cell Lines and Tumors
The observation that human tumor cell lines have reduced PlGF expression, but maintained the ability to support PlGF promoter activity, suggested that reduced expression might be due to the epigenetic modification of the PlGF gene, such as promoter methylation or histone deacetylation. We subsequently hypothesized that promoter hypermethylation might be involved in the down-regulation of PlGF in human tumors. First, we analyzed the methylation status of PlGF in lung and colon carcinoma cell lines and tumor tissues using methylation-specific PCR (MSP). Genomic DNA was isolated from six lung carcinoma cell lines and six colon carcinoma cell lines and modified by sodium bisulfite. MSP analysis of the methylation state of the Plgf promoter indicated aberrant DNA methylation in all of the lung and colon carcinoma cell lines (Fig. 4A
). BeWo cell, which express high levels of PlGF (Fig. 1B), was used as control. In contrast, the VEGF promoter was not methylated as analyzed by MSP. The human umbilical vascular endothelial cell, which does not express VEGF and has been reported to involve promoter methylation for VEGF transcriptional regulation, was used as control (25).
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FIGURE 4. Methylation of Plgf promoter in human lung and colon carcinoma cell lines. A. MSP of Plgf and Vegf promoter in human lung and colon carcinoma cell lines. M and U, amplification using methylated and unmethylated sequence-specific primers, respectively. B. Results of sequence analysis of bisulfite-treated genomic DNA from A549, NCI-H1299, HCT116, and LS174T cells. Genomic DNA from five individual clones from each cell sample was sequenced; the methylation status is summarized. Row, one sequenced clone. Circle, a CpG dinucleotide: •, methylated;  , unmethylated.
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Next, genomic DNA from A549, NCI-H1299, HCT116, and LS174T were modified by bisulfite treatment. PlGF genomic DNA (–197/+25) was then PCR amplified and subcloned. Unbiased primers were chosen based on their lack of CpG dinucleotide sites. PCR products were purified and cloned into the pCR4-TOPO vector and sequenced. Five clones from each cell sample were selected for sequence analysis, and 37 CpG dinucleotides in the PlGF promoter were examined. The methylation status of the PlGF promoter from all cells is summarized in Fig. 4B. These data show that the PlGF gene is hypermethylated in human lung and colon carcinoma cell lines.
To investigate whether aberrant methylation also occurred in vivo, we analyzed the methylation status of the Plgf promoter in 22 lung and 11 colorectal tumor samples using MSP. Methylation of the Plgf promoter was detected in all of the lung (Fig. 5A
) and colon carcinoma samples, but not in normal lung and colon genomic DNA (Fig. 5B). U primer that amplifies the unmethylated DNA also gave amplification products. This may be due to the fact that these studies were done with whole tumor genomic DNA, which also includes genomic DNA from normal tissues.
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FIGURE 5. Methylation status of Plgf promoter in lung and colorectal cancer patient samples. A. MSP of Plgf promoter in representative samples of lung carcinoma. B. MSP of Plgf promoter in representative samples of colorectal carcinoma. 1 to 10, genomic DNA from lung or colon sample 1 to 10; N, normal genomic DNA from the corresponding tissue.
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PlGF Gene Expression Is Greatly Increased by Demethylation
To study whether epigenetic alterations of the PlGF gene contribute to the reduced PlGF mRNA expression, we treated two lung cancer cell lines and two colon carcinoma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-Aza-dC) for 96 h, and then PlGF mRNA expression levels were analyzed by Northern blot. If promoter hypermethylation plays a role in the reduction of PlGF expression, treatment of these cells with methylation inhibitors should increase or restore PlGF mRNA expression. As shown in Fig. 6A
, the effective up-regulation of PlGF mRNA expression was achieved in all four cell lines treated with 5-Aza-dC. Furthermore, ELISA analysis revealed the presence of PlGF protein in conditioned medium collected from these four cells after 96 h treatment with 5-Aza-dC (Fig. 6B). These data indicate that promoter hypermethylation can play a role in the reduced expression of PlGF in these tumor cell lines.
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FIGURE 6. Restoration of PlGF gene expression after treatment with demethylating agents. Two lung carcinoma cells, A549 and NCI-H1299, and two colon carcinoma cells, HCT116 and LS174T, were plated in a 10-cm dish and then treated with either DMSO (control) or 5 µmol/L 5-Aza-dC for 96 h. A. Northern blot analysis of PlGF mRNA expression. B. PlGF protein expression was analyzed by ELISA. C. Tumor cells were incubated in the presence or absence of 0.5 µmol/L TSA for 24 h, and then mRNA was isolated for Northern blot analysis of PlGF mRNA expression.
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To examine the role histone deacetylase activity plays in the inhibition of the Plgf gene in lung and colon carcinoma, cells were treated with the histone deacetylase inhibitor trichostatin A (TSA) alone and in combination with 5-Aza-dC. As analyzed by Northern blot analysis (Fig. 6C), treatment with TSA alone failed to restore the expression of PlGF mRNA in all cell lines. Furthermore, treatment with TSA after 5-Aza-dC treatment did not result in enhanced levels of PlGF mRNA expression in any of the cell lines either (data not shown).
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Discussion
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In this study, we observed that PlGF expression is low in human lung and colon tumors and in cell lines. By combining the results from cDNA microarray, immunohistochemistry, and MSP, we have reported, for the first time, the low expression level of PlGF in human lung and colorectal cancers correlated with Plgf promoter hypermethylation. Furthermore, we showed that the DNA demethylating agent restored PlGF expression at both mRNA and protein levels in the human lung and colon carcinoma cell lines.
We choose to study the status of PlGF promoter methylation status in human lung and colorectal tumors for two reasons. First, whereas the molecular mechanisms that regulate the expression of its family member, VEGF, have been well documented (26, 27), the regulatory mechanism of PlGF expression is not known. One study showed that PlGF expression was induced in mouse fibroblast by hypoxia (28), other studies showed PlGF expression in trophoblast decreased under hypoxia (29). Virtually nothing is known of what regulates PlGF expression in human tumor cells. Our preliminary study showed that PlGF expression level was low in lung and colorectal tumor tissues, and this prompted us to study the mechanisms that inhibited the expression of PlGF in these two tumor types. Second, the role of PlGF in tumor angiogenesis and progression is controversial; some published studies suggest that PlGF inhibits tumor angiogenesis and growth (8, 9), indicating that PlGF functions as a tumor suppressor gene. Aberrant promoter methylation has been shown to be a common mechanism that regulates tumor suppressors in non–small cell lung cancer and colorectal cancer. These include genes involved in cell cycle regulation and angiogenesis (cyclooxygenase-2, p14, and p16), DNA repair or protection (hMLH1, MGMT, and GSTP1), signal transduction (APC and RASS-F1A), and those related to metastasis and invasion (E-cadherin and TIMP-3; refs. 23, 24). Thus, we hypothesized that promoter hypermethylation contributes to the suppression of PlGF in human lung and colon carcinomas.
In our study, elevated VEGF level was observed in tumor tissues. On the contrary, PlGF expression level is low in tumor tissues compared with the corresponding normal tissue. This could be because most of the tumor tissues included in our study are from early-stage patients. This is in accordance with two recently published studies showing that PlGF expression correlates with microvessel density (19) and inversely correlates with disease progression and patient survival in non–small cell lung cancer and colorectal cancer (18).
VEGF has been well documented to be essential for normal vascular development and promotes angiogenesis in a variety of in vitro and in vivo settings by modulating proliferation, permeability, and survival. However, the role of PlGF in tumor angiogenesis is controversial. Three studies from Carmeliet et al. (5-7) showed that PlGF enhances tumor angiogenesis; on the contrary, studies from Cao et al. (8, 9) showed that PlGF inhibits the angiogenic effect of VEGF by forming inactive PlGF/VEGF heterodimers. Our recent studies showed that the overexpression of PlGF inhibits human lung and colon carcinoma growth, angiogenesis, and metastasis in orthotopical mouse models, which indicates that PlGF functions as a tumor suppressor gene in the progression of human lung and colorectal carcinomas (10). It is not uncommon to find genes with disparate functions within the same family. Prominent examples can be found in the BCL2 family of proteins, in which members can mediate either proapoptotic or antiapoptotic effects (30).
The chromatin structure is a dominant force in methylation-associated gene silencing. The methyl-CpG–binding protein MeCP2 has been shown to recruit histone deacetylase activity to in vitro methylated promoters, indicating that these two processes are linked. Thus, it may be necessary to simultaneously block both DNA methylation and histone deacetylation to achieve maximal reactivation of genes silenced by methylation (31-33). TSA has been shown to act in restoring the expression of densely methylated genes (33). However, in our study, histone deacetylase inhibitors did not restore PlGF expression, nor did it have any additive effect with inhibiting methyltransferase. Thus, histone deacetylation does not seem to play a significant role in the inhibition of PlGF in human lung and colon carcinoma cell lines.
Studies from phase I trial of bevacizumab in rectal patients showed increased plasma PlGF levels after VEGF blockade (20). A more recent study of phase II clinical trial of SU11248 in metastatic renal cell carcinoma patients also reported elevated plasma PlGF levels after inhibition of VEGF receptor and platelet-derived growth factor receptor (21). The elevated PlGF level may indicate a role of PlGF in tumor escape therapy, thus warranting the exploration of anti-PlGF strategies. However, the mechanisms that made PlGF unregulated by these agents need further investigation.
This report is the first to show that low PlGF expression in human tumor tissues and cell lines is correlated with Plgf promoter hypermethylation. Treatment with the demethylating agent restored PlGF expression in tumor cell lines, confirming that the low expression of this gene in cell lines was a result of methylation. Additional studies are clearly needed to further understand potential mechanisms that regulate PlGF expression and to evaluate PlGF as a potential target in cancer therapy.
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Materials and Methods
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Cell Culture
Lung carcinoma cell lines, A549, NCI-H1299, NCI-H358, NCI-H526, NCI-H1395, NCI-H69; colon carcinoma cell lines, Lovo, HCT116, HT-29, SW48, SW620, and human trophoblast cell line, BeWo, were obtained from the American Type Culture Collection (ATCC). The tumor cell line was maintained as instructed by ATCC.
Expression Analysis Using the Tumor Tissue Array
The cancer profiling array consists of 154 cDNA pairs, synthesized from human tumorigenic and corresponding normal tissue (http://www.clontech.com/techinfo/manuals/pdf/pt3578-1.pdf). Each pair was independently normalized based on the expression of three housekeeping genes and was immobilized in separate dots. Plgf cDNA was radiolabeled using Random Prime Labeling kit (Amersham Pharmacia Biotech). Hybridization of the labeled probe was done as instructed by the manufacturer.
Tissue Specimens
The 22 lung carcinoma– and 11 colon carcinoma–archived samples used in this study were obtained from cancer patients by surgical resection in the Massachusetts General Hospital (Boston, MA) under an Institutional Review Board–approved protocol. None of the patients were treated with preoperative chemotherapy or radiation therapy. Tumor specimens composed of at least 80% carcinoma cells were chosen for molecular analysis. Tumors were graded according to the American Joint Committee on Cancer and staged according to the 1997 tumor-node-metastasis (TNM) system. The 22 lung carcinomas include 13 adenocarcinomas, 7 squamous cell carcinomas, and 2 bronchioalveolar carcinomas. The TNM staging consisted mainly of three groups (I, n = 9; II, n = 10; III, n = 3). The 11 colorectal adenocarcinoma samples were of stage III. Specimens of normal lung and colorectal tissues distant to the tumor tissue were examined as controls.
Northern Blot Analysis
Northern blot was done as described previously (34). cDNA probes were synthesized by PCR using primers for Plgf: 5'-TCGTCAGAGGTGGAAGTGGT-3'; 5'-CTTCATCTTCTCCCGCAGAG; and for ß-actin: 5'-TGTATGCCTCTGGTCGTACC-3'.
Plasmid Constructs
The 3.2-kb human PlGF promoter (GenBank NT_026437) was obtained by PCR. Human placenta genomic DNA (Sigma) was used as template. The PCR product was sequenced and confirmed to be identical to the published sequence of PlGF promoter. The PCR product was digested with SacI and HindIII and subcloned into pGL3-Basic (Promega).
Transient Transfections and Reporter Gene Assays
Transient transfection and reporter gene assays were done as described previously (34). The cells were cotransfected with 20 µg pGL3-Basic or pGL3-plgf and 2 µg of pRL-TK/plate using the LipofectAMINE 2000 (Invitrogen). The transfected cells were lysed 24 h after transfection using the Dual Luciferase Assay System (Promega). The light intensity was measured on 20 µL of cell lysates using a luminometer.
Measurement of Protein Production by ELISA
Protein production was analyzed by ELISA using the Quantikine PlGF and VEGF ELISA kit (R&D Systems) according to the manufacturer's instructions.
Immunohistochemistry
PlGF (1:50, Santa Cruz Biotechnology) staining was carried out on tissue sections of formalin-fixed, paraffin-embedded human lung and colorectal carcinoma tumor tissues. This antibody stains PlGF specifically and does not cross-react with VEGF. As positive controls, tissue from BeWo trophoblast xenografts, which expresses a high level of PlGF, was included in each staining run. As negative control, the sections were stained with nonspecific immunoglobulin G under the same conditions.
DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was isolated from human tumor cell lines and archived tumor specimens using a Dneasy Tissue Kit (Qiagen). Sodium bisulfite modification of genomic DNA was conducted using the CpGenome DNA kit according to the manufacturer's instructions (Chemicon). After bisulfite modification, the PlGF promoter sequence was amplified by PCR using the following primers: U primers, 5'-GATGTTTTTATTTTAGGTGATTGT-3' and 5'-TCCCTTCTAAAACTACAACCAAT-3', was designed for the unmethylated sequence of the Plgf promoter region. M primers, 5'-GAGATACGGAGTTTGGTACG-3' and 5'-AACTTAAACGCGAAAAACGA-3', were used for the methylated sequence. Bisulfite-specific primers were designed using Primer3 Software.1 Methylated DNA sequences were only amplified by M primers but not by unmethylated specific primers (U primers). For sequencing, PlGF promoter was amplified by PCR using the following primers (forward, 5'-AATTTGGTGTAGTTAGGTGA-3', –197/–177, and reverse, 5'-CATCTCCCTAACCCAAATTA-3', +5/+25). PCR products were subcloned into the pCR4-TOPO vectors (Invitrogen). Plasmids from five clones of each cell line were sequenced. Standard settings of the BLAST 2 sequences alignment tool were used for comparison of human Plgf promoter sequences.
5-Aza-dC and TSA Treatment
Cells were plated in a 10-cm dish 24 h before treatment. 5-Aza-dC (5 µmol/L) (Sigma) was added to the fresh medium, and the cells were harvested after 4 days. TSA (0.5 µmol/L) was added to the fresh medium, and the cells were harvested after 24 h.
Statistical Analysis
The significance of the data was analyzed by the Student's t test (two tailed).
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Acknowledgements
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We thank Chelsea J. Swandal and Song Xu for their technical support.
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Notes
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Grant support: Program Project Grant P01-CA80124, R01-CA85140, and U01-CA84301 from the National Cancer Institute (to R.K. Jain).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 http://frodo.wi.mit.edu/primer3 
Received 5/17/06;
revised 4/30/07;
accepted 6/ 8/07.
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Abstract
Introduction
