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Molecular Biology and Genetics |
and ß in Human Cancers1
Lovelace Respiratory Research Institute, Lung Cancer Program, Albuquerque, New Mexico 87108 [W. A. P., K. P. C., M. J. G., S. A. W., S. A. B.]; First Department of Internal Medicine, Sapporo Medical University, Sapporo 060-8543, Japan [M. T.]; Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center, Majadahonda 28220, Spain [M. E.]; Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131 [N. J.]; and Johns Hopkins University Medical Institutions, The Johns Hopkins Comprehensive Cancer Center, Baltimore, Maryland 21231 [S. B. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß genes. These genes, located on chromosome 9p13, are transcribed from two distinct promoters and form two alternative first exons that are subsequently spliced to the common exons 2–10. The resulting splice variants encode two distinct transcription factors important in cell differentiation and embryonic development. Examination of the methylation status of each gene using methylation-specific PCR revealed that both genes are methylated in 
65% of breast and lung tumors. Bisulfite sequencing revealed dense methylation patterns within each 5'CpG island, strongly correlating with transcriptional silencing. Expression in cell lines with dense methylation of either the PAX5 or ß promoter region was restored after treatment with the demethylating agent 5-Aza-2'-deoxycytidine. The PAX5 ß gene encodes for the transcription factor B cell-specific activating protein that, in turn, directly regulates CD19, a gene shown to negatively control cell growth. A strong association was observed between PAX5 ß methylation and loss of expression of the CD19 gene demonstrating that inactivation of the PAX5 ß gene likely contributes to neoplastic development by inhibiting growth regulation through effects on CD19 gene expression. Recent studies have demonstrated the importance of PAX5 gene alterations in human cancer. Our results are the first to identify aberrant promoter methylation as a common mechanism for dysregulation of these genes in solid tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several cancer genome-wide screening approaches have been developed to identify genes inactivated by promoter hypermethylation. These approaches include methylation-sensitive arbitrarily primed PCR (8 , 9) , restriction landmark genomic scanning (10) , CpG microarrays (11 , 12) , methyl-CpG binding domain chromatography (13) , and MCA3 coupled with RDA (14) . The MCA/RDA approach has identified several methylated genes involved in colorectal (15 , 16) and pancreatic cancers (17) . MCA/RDA is a PCR/subtraction hybridization-based assay that allows for the rapid amplification and selection of densely methylated CpG-rich regions ranging in size from 200 bp to 2 kb. The purpose of the current study was to use the MCA/RDA technique to identify novel genes inactivated by promoter hypermethylation in breast cancer and to determine the commonality for gene inactivation in lung cancer, another solid tumor.
Using amplicons from the breast cancer cell line MCF7 as the tester, and amplicons from normal breast tissue as the driver, one region identified was the 5' untranslated region and exon 1 of the transcription factor PAX5. The PAX5 gene plays an important role in cell differentiation and embryonic development, and is located on chromosome 9p13 (18)
. This locus is associated frequently with chromosomal translocations and contains two distinct promoters resulting in two alternative 5' exons ( and ß) that are spliced to common coding sequences of exons 2–10 (19)
. The current studies demonstrate that the PAX5 and ß genes are both frequent targets for aberrant methylation in tumor cell lines, as well as primary tumors from breast and lung.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MCA/RDA.
MCA/RDA was performed as described by Toyota et al. (14)
. Briefly, 5 µg of DNA from the cell line MCF7 was used as the tester, and a mixture of DNA from normal breast tissue of five women (1 µg each) was used as the driver. MCA amplicons were produced using adaptors, and two rounds of competitive hybridizations were performed. The resulting RDA products were digested with the restriction endonuclease XmaI (New England Biolabs) and subsequently cloned into pBluescript KS+.
DNA Sequencing and Analysis.
Plasmid DNA containing the RDA products was prepared using the QIAprep Spin Miniprep according to the manufacturer’s instructions (Qiagen). Virco (Cambridge, United Kingdom) analyzed the sequences using an automated DNA sequencer (Applied Biosystems). Sequence homologies were determined using the Blast program of the National Center for Biotechnology Information.4
MSP and Bisulfite Sequencing.
DNA was isolated by standard phenol-chloroform extraction and ethanol precipitation. Genomic DNA was modified by treatment with sodium bisulfite that converts only unmethylated cytosines to uracil. The methylation status of the PAX5 and ß genes was determined using a nested, two-stage method described previously (21)
. Primer sequences used in the stage-1 amplification of each gene are as follows: -Forward, (5'-GGGTTTGTATATGGAGATGTTATAGG-3'); -Reverse, (5'-CAACATCACAAAATATCCCCAAACAC3'); ß-Forward, (5'-AGTTTGTGGGTTGTTTAGTTAATGG-3'); and ß-Reverse, (5'-CAAAAAATCCCAACCACCAAAACC-3'). All of the PCR amplifications were performed using a Biometra T3 thermocycler and Taq Gold polymerase (Perkin-Elmer). The cycling parameters for stage-1 were as follows: 94°C for 10 min; then 40 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; and a 5-min final extension at 72°C. Stage-1 ß conditions were identical except the annealing temperature was reduced to 54°C, and all of the cycling times were performed for 30 s. The size of the stage-1 and ß PCR products was 389 and 328 bp, respectively.
Primer sequences used in the stage-2 amplification of each gene are as follows: -BSM1, (5'-ATAAAAGTTTGGGGCGGCGC-3'); -BSM2, (5'-GCGCCCCCAACGCGCCG-3'); ß-BSM1, (5'-GAGTTGAGTTTCGGGCGGC-3'); and ß-BSM3, (5'-GCCGCCGCCGCCGTCG-3'). The cycling parameters for stage-2 were as follows: 94°C for 10 min; then 40 cycles of 94°C for 15 s, 66°C for 15 s, and 72°C for 15 s; and a 5-min final extension at 72°C. Stage-2 ß conditions were identical except the annealing temperature was decreased to 64°C. The size of the stage 2- and ß PCR products was 166 and 124 bp, respectively. DNA isolated from cell lines MDA-MB-231 and NCI-H2009 served as positive and negative controls, respectively, for both genes. All of the assays were conducted in at least duplicate, and positive methylation was confirmed by restriction digestion with BstUI or by DNA sequencing.
Bisulfite-modified DNA from the T47D and MCF-7 cell lines was amplified using the stage-1 primers for the PAX5 and ß genes. Stage-1 and methylation-specific stage-2 primers were used to amplify modified DNA from three breast tumors shown to be methylated for the PAX5 or ß gene. The PCR products were ligated into the PCR II vector using the TA cloning kit (Invitrogen, San Diego, CA). Three to five clones from each sample were sequenced.
DAC Treatment and RT-PCR.
Re-expression studies for PAX5 and ß were performed using three breast cancer cell lines (MCF7, MDA-MB-231, and T47D) and one lung cancer cell line (Calu6). Cell lines were treated for 3 days in culture medium with 1 µM DAC (Sigma Chemical Co.). Total RNA was prepared using TRIzol (Life Technologies, Inc.), and 3 µg aliquots were reverse-transcribed using the Superscript kit (Life Technologies, Inc.). The expression of the PAX5 and ß transcripts was determined by RT-PCR using the exon 1 forward primer (5'-CCTGTCCATTCCATCAAGTCCTG-3') and the exon 1ß forward primer (5'-CCCGATG GAAATACACTGTAAGCAC-3') with an exon 2 reverse primer (5'-TTTTGCTGACA CAACCATGGCTGAC-3'). PCR amplification was performed at 94°C for 10 min; then 40 cycles of 94°C for 30 s, 64°C for 30 s, and 72°C for 30 s; and a 5-min final extension at 72°C.
The expression of the CD19 gene was examined in 6 bronchial epithelial cell lines, in 2 breast cancer cell lines (SKBR3 and MDA-231), and in 3 lung cancer cell lines (NCI-H23, A549, and Calu6). Cell lines were treated with DAC and RNA isolated as described above. CD19 expression was determined after RT-PCR using the forward primer (5'-CTCAGCCAGGACCTCACCATG-3') and reverse primer (5'-CATAGTACTGGCCGAGCA GTGA-3'). PCR amplification was performed at 94°C for 10 min; then 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and a 5-min final extension at 72°C. ß-Actin was also amplified as a control for RNA integrity. PCR amplification was performed at 94°C for 10 min; then 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and a 5-min final extension at 72°C. PCR products for all of the genes were analyzed on a 3% agarose gel containing ethidium bromide and visualized under UV illumination.
Data Analysis.
The proportion of tumors positive for methylation of PAX5 or ß was compared among cancer types with the Fisher’s exact test. The association between PAX5 and ß methylation for each cancer type was also assessed by the Fisher’s exact test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß Hypermethylation by MCA. and 1 ß) that are spliced to common exons 2–10 resulting in the translation of two unique proteins because of a frameshift (Fig. 1
; Ref. 19
). This allowed us to address coordinate aberrant regulation of two genes that share common exons, a scenario identical to the p16INK4a and p14ARF genes. The region identified by the MCA/RDA approach was localized in the 5' flanking region and exon 1, the site for most CpG islands. Inspection of the PAX5 and ß promoters revealed that each region is representative of a CpG island (22
, 23)
. The GC content was 0.68 and 0.6 with a CpG:GpC ratio of 0.70 and 0.73; 53 and 162 CpG sites were found in a 520-bp and 1780-kb region, respectively.
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and ß Genes. and ß genes. After bisulfite modification, PCR products were produced using stage-1 primers that do not discriminate between methylated and unmethylated alleles. Methylated alleles were detected in the PAX5 (389 bp) and ß (328 bp) products after digestion with the restriction enzyme BSTU1, which specifically cleaves at CGCG sites that are retained after bisulfite modification because of the presence of methylated CpGs. Complete digestion of the PCR products indicative for methylation of the PAX5 gene was observed in 7 breast and 5 lung cancer cell lines. The PAX5 PCR product was partially digested (10–80%) for 2 breast and lung cancer cell lines. Similarly, the PAX5 ß gene was completely digested in 5 breast and 3 lung cancer cell lines. The PAX5 ß PCR product was also partially digested (10–80%) for 1 lung and 2 breast cancer cell lines. PCR products from normal lymphocytes remained undigested, indicating a lack of methylation (data not shown).
The frequency of PAX5 and ß methylation was then characterized in a panel of primary tumors (breast and lung) using our two-stage MSP approach (21)
. The results are summarized in Table 1
, and a representative MSP analysis is shown in Fig. 2
. In both breast and lung tumors, the frequency of PAX5 methylation approximated 70% and tended to be greater than that observed for the PAX5 ß gene (54%). Methylation of the ß gene was strongly associated (P = 0.05) with methylation of the gene in lung tumors and cell lines suggesting that some selective advantage to target both genes for inactivation in this cancer type may exist (Table 1)
. This association was not observed in breast tumors.
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and ß genes, respectively. Eleven lung tumor-derived cell lines were analyzed by MSP, and 82 and 55% were methylated for the PAX5 and ß genes, respectively.
Methylation of the PAX5 genes was also examined in tissue adjacent to the tumors using our sensitive two-stage MSP approach to determine the extent of the tumor spread within the resected tissue. PAX5 methylation was seen in 4 of 8 and 6 of 10 adjacent tissues from breast and squamous cell tumor cases, respectively (Table 1)
. PAX5 ß methylation was not seen in any adjacent tissue from breast cancers, but was seen in 5 of 10 adjacent tissue from squamous cell tumor cases. Methylation was only detected in adjacent tissue from breast tumors that were also positive for methylation. However, one squamous cell tumor negative for PAX5 and another tumor negative for ß had faint methylation signals in their respective adjacent lung tissue. The detection of PAX5 methylation in tissue adjacent to breast and squamous cell carcinomas probably reflects tumor spread, because 80% of the cases evaluated had lymph nodes positive for malignancy or field cancerization commonly seen in bronchial epithelium from smokers (20)
. The detection of epigenetic and genetic changes in tissue adjacent to tumors is quite common and has important implication for predicting disease recurrence. For example, patients with squamous cell carcinoma of the head and neck with cytologically negative tumor margins but positive for p53 mutation had a substantial risk for local recurrence (24
, 25)
. To determine whether methylation of the PAX5 genes was specific to cancer, we examined bronchial epithelial cells from 35 cancer-free heavy smokers and peripheral blood mononuclear cells from 20 never-smokers. Methylation of the PAX5 and ß genes was detected in bronchial epithelial cells from only 1 of 35 smokers (Table 1)
. Neither PAX gene was methylated by MSP in any blood lymphocytes from never-smokers.
Re-Expression of the PAX5 and ß Genes after DAC Treatment.
The expression of the PAX5 and ß transcripts was determined by RT-PCR on cDNA from three normal lung tissues adjacent to squamous cell tumors that were negative for PAX5 methylation. Expression of both transcripts was readily detected in all three of the adjacent lung tissues (Fig. 3A)
. RT-PCR was also conducted on cDNA from three breast cancer cell lines (MCF7, MDA-MB 231, and T47D) and one lung cancer cell line (Calu6) to determine the relationship between methylation of the PAX5 and ß genes, and transcription. COBRA analysis demonstrated complete digestion for the PAX5 gene in the MDA-MB-231 and T47D cell lines and partial digestion for MCF7 (20%) and Calu6 (80%). For the PAX5 ß gene, complete digestion was seen in the MCF7, MDA-MB 231, and Calu6 cell lines, but no digestion in T47D. These methylation changes were corroborated by MSP (Fig. 3B)
. PAX5 transcript was abundant in MCF7 and to a lesser extent present in Calu6, whereas no transcript was present in the MDA-MB 231 and T47D cell lines (Fig. 3C)
. Treatment of these cell lines with 1 µM DAC, an inhibitor of DNA methyltransferase, increased expression in Calu6 and restored expression in MDA-MB231 and T47D. PAX5 ß transcript was only detected in T47D; however, treatment with DAC restored expression in the other three cell lines. Treatment with DAC did not affect the expression of the housekeeping gene ß-actin.
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and ß Promoters. and ß promoter regions amplified by the MSP primers was determined for the MCF-7 and T47D cell lines, and three primary breast tumors. The PCR primers used for the COBRA analysis were also used to amplify the modified DNA from the cell lines, whereas methylation-specific primers were used to detect methylated sequences in the primary tumors where contaminating stromal and inflammatory cells are present. Sixteen and 14 CpG sites spanning the 116- and 86-bp regions between the MSP primers for the PAX5 and ß sequence, respectively, were evaluated to compare methylation density among cell lines and tumors. The sequencing results corroborated the expression studies with 78% of sites methylated within the PAX5 promoter in the T47D cell line, and 100% of sites methylated within the PAX5 ß promoter in the MCF-7 cell line (Figs. 3
and 4)
. The CpG methylation density in all three of the primary tumors was very similar to the patterns observed in the methylated cell lines for each gene, consistent with these regions being important sites for methylation and silencing of the PAX5 and ß genes (Fig. 4)
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. In addition, CD19 expression was observed in cell lines SKBR3 and NCI-H23 that lack methylation of the PAX5 ß gene (Fig. 5B)
. In contrast, no CD19 expression was detected in the Calu6, MDA-MB-231, and A549 cell lines, all of which are methylated for PAX 5 ß. Treatment of these cell lines with DAC restored expression of the CD19 gene (Fig. 5B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß genes as common targets for inactivation by aberrant promoter hypermethylation in lung and breast tumors. Both genes exhibited homogeneous and dense methylation patterns that correlated with transcription loss. Silencing of the PAX5 ß gene was associated directly with loss of CD19 gene expression in lung and breast cancer cells. A similar finding was observed in murine myeloma cells (30)
. Thus, the tight regulation of CD19 gene transcription by the PAX5 ß-encoded BSAP protein likely contributes to the selective growth advantage displayed by both solid and liquid tumors. CD19 antibodies provided a positive proliferation signal for early precursor B cells indicating that CD19 is involved in the negative control of proliferation (31)
.
Previous studies using MCA and RDA identified the PAX6 gene that is involved in eye, nose, pancreas, and brain development as a common target for aberrant methylation in colonic mucosa during aging (14
, 15)
. The CpG island identified was localized within an enhancer present in the 5' region of the PAX6 gene. A CpG island within the exon 5 coding region of the PAX6 gene was also hypermethylated in bladder and colon tumors (32)
. Interestingly, whereas methylation of the promoter region and exon 5 was common in cell lines, methylation in primary tumors was largely confined to exon 5 and did not affect gene transcription. This was not the case for the PAX5 and ß genes where dense methylation was seen in both promoter regions in derived cell lines, and primary lung and breast cancers that correlated directly with loss of transcription. The difference seen in primary tumors for silencing the PAX5 and 6 genes may be linked to their functions in the respective tissues. No expression of the PAX6 gene was seen in normal colonic mucosa, thereby negating any selective advantage for methylation of the promoter region, whereas we and others observed abundant expression of both PAX5 and CD19 transcripts in normal lung tissue and bronchial epithelial cells (33)
.
A critical role for the PAX5 gene in development is evident from knockout studies in mice. Homozygous mutant mice were born alive, but their growth was retarded, and they usually died within 3 weeks. Developmental defects include alteration of the posterior midbrain morphogenesis and a complete arrest of B-cell development at early precursor stages (34)
. Gene targets regulated directly or indirectly by the PAX5 ß gene have been identified (27)
. For example, the BSAP protein can regulate p53 and IgHC genes, in addition to CD19 (28
, 35)
. The BSAP protein can bind directly to a sequence in the 5' region of the p53 gene to inhibit expression, and dysregulation of this process has been correlated with p53 protein levels in astrocytomas (28
, 35)
. Whereas this paradigm appears limited to the brain, it does speak to a critical role for the BSAP transcription factor in modulating proteins involved in basal cellular activity. Our studies now indicate that the BSAP protein directly regulates CD19 expression in both lung and breast tumors. There is precedent for inactivating genes that code for transcription factor binding proteins by promoter hypermethylation. Hypermethylation in cancer (HIC-1) is a zinc-finger transcription factor gene that is commonly expressed in normal tissues, but inactivated by promoter hypermethylation in lung, breast, colon, and hematopoietic tumors (36
, 37)
. Similar to the PAX5 gene, HIC-1 is also important in development with knockout mice dying perinatally and exhibiting gross developmental defects involving the brain, cleft palate, and limbs (38)
. As downstream targets are identified for the PAX5 protein, the impact of silencing this gene on normal cellular function will be realized.
Our recent studies suggest that changes in gene-specific methylation could serve as intermediate biomarkers for cancer detection, risk assessment, and monitoring disease in sputum and blood (21
, 39)
. Our group has focused largely on lung cancer where the aberrant methylation of the p16 and/or O6-methylguanine-DNA methyltransferase promoters was detected in DNA from sputum of patients with squamous cell carcinoma up to 3 years before clinical diagnosis (21)
. The development of risk models for predicting lung cancer through marker detection in biological fluids such as sputum will necessitate the conduct of longitudinal studies in high-risk populations and the development of a panel of markers that are inactivated throughout the progressive phases of cancer development. The fact that methylation of the PAX5 and ß genes was found only in tumors and surrounding tissue, and only rarely in normal epithelial cells, albeit from a heavy smoker, supports their evaluation as intermediate markers in our model. Other groups have also demonstrated the utility of plasma or serum for the detection of circulating aberrantly methylated DNA in people with colorectal, head and neck, lung, and breast cancers (39, 40, 41)
. Thus, the inclusion of the PAX5 genes into molecular marker panels for lung and breast cancer may improve the sensitivity and specificity for developing risk models for detecting these cancers through analysis of sputum and blood in high-risk subjects.
Our studies identify a new class of genes, those that code for transcription factor binding proteins, which are methylated at high prevalence in the two most common human cancers. The silencing of the PAX5 ß gene is linked functionally to CD19, a gene already established as a key regulator of the cell cycle. This link implicates the targeting of another pathway that controls the cell cycle for inactivation in liquid and now solid tumors.
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FOOTNOTES |
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1 Supported by Department of Defense Breast Cancer Initiative DAMD17-99-1-9258, RO1-ES-08801, P50-CA-58184, and the University of New Mexico Cancer Center Sequencing Shared Facility. 
2 To whom requests for reprints should be addressed, at Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108-5127. Phone: (505) 348-9465; Fax: (505) 348-4990; E-mail: sbelinsk{at}LRRI.org
3 The abbreviations used are: MCA, methylated CpG island amplification; RDA, representational difference analysis; DAC, 5-aza-2'-deoxycytidine; RT-PCR, reverse transcription-PCR; MSP, methylation-specific PCR; BSAP, B-cell-specific activating protein; COBRA, combined bisulfite restriction analysis.
4 Internet address: http://www.ncbi.nlm.nih.gov/BLAST/.
Received 1/22/03. Accepted 5/28/03.
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REFERENCES |
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and 
indicate that the CpG site is unmethylated or methylated, respectively. Each row represents one clone.