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Transcriptional Silencing of the p73 Gene in Acute Lymphoblastic Leukemia and Burkitt’s Lymphoma Is Associated with 5' CpG Island Methylation¹

The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [P. G. C., M. E., S. B. B., J. G. H.]; Academic Medical Center, Department of Human Genetics-M1, 1105 AZ Amsterdam, the Netherlands [M. M. v. N.]; and Pediatric Hematology and Oncology, Rainbow Babies and Children’s Hospital [S. J. K., N. C.], and the Department of Human Genetics [S. J. K.], Case Western Reserve University, Cleveland, Ohio 44106

	ABSTRACT

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he p73 gene is located on 1p36.2-3, a region that is frequently deleted in human cancer. Because p73 encodes for a protein that is both structurally and functionally homologous to the p53 protein, p73 has been postulated to be a candidate tumor suppressor gene. To date, however, mutations of p73 have not been found. To study methylation of the p73 5'CpG island, a human bacterial artificial chromosome clone containing exon 1 and the 5' region of p73 was isolated. There was no evidence for p73 exon 1 methylation in normal tissues. In contrast, p73 was aberrantly methylated in approximately 30% of primary acute lymphoblastic leukemias (ALLs) and Burkitt’s lymphomas. There was no evidence for methylation in any other types of hematological malignancies or solid tumors examined. In both leukemia cell lines and primary ALLs, methylation was associated with transcriptional loss of p73 by reverse transcription-PCR. We used single-strand conformational polymorphisms to screen for point mutations in a series of primary ALLs and found no mutations leading to a change in protein structure. Our results show that methylation of p73 is a frequent event in specific types of hematological malignancies and suggest that epigenetic silencing of p73 could have important consequences for cell-cycle regulation.

	Introduction

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p73, a gene with significant homology to p53, was recently cloned and localized to chromosome 1p36.2-3 (1) . The p73 protein is structurally similar to the p53 protein within its DNA-binding, transactivating, and oligomizeration domains. The p73 protein also shares some functional characteristics with p53, including the ability to promote apoptosis when overexpressed in vitro and up-regulate p53 responsive genes involved in cell-cycle control such as p21 (2) . Loss of heterozygosity of 1p is common in human neoplasias including neuroblastoma, melanoma, hepatocellular carcinoma, colorectal carcinoma, and breast cancer (3) , and it has been postulated that p73 is a candidate tumor suppressor gene (1) . To date, however, somatic mutations of p73 have not been found in neural, colorectal, lung, or prostate tumors (4, 5, 6, 7, 8) .

Genetic analysis of neuroblastomas with deletions of 1p36 show loss predominantly of the maternal allele, suggesting that the tumor suppressor gene(s) in this region is imprinted. In support of this finding, p73 seems to be monoallelically expressed in neuroblastoma cell lines, normal lymphocytes, and normal kidney (1 , 9) , which raises the possibility that inactivation of p73 would require only a single event leading to preferential loss of the expressed allele. In normal tissues, 5' cytosine methylation of CpG dinucleotides located within promoter CpG islands has been associated with transcriptional silencing of imprinted genes and genes located on the transcriptionally silent X chromosome of the female (10) . In the development of cancer, this epigenetic process occurs aberrantly and has been reported to inactivate a number of tumor suppressor genes and genes that preserve normal cellular function, including O⁶ methylguanine-DNA methyltransferase and tissue inhibitor of metalloproteinase-3 (11 , 12) . DNA methylation alters the binding of transcription factors to gene-regulatory regions and is associated with a repressive chromatin structure (13) . Because of the interesting possibility that p73 is both an imprinted gene and a tumor suppressor, we have studied the methylation status of the p73 CpG island in both normal tissues and cancers.

In this study, we report evidence for aberrant hypermethylation of the p73 promoter in ALLs⁴ and Burkitt’s lymphomas. This finding led us to screen for p73 mutations in a series of ALLs.

	Materials and Methods

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Samples, Cell Lines, and Culture Conditions.
The majority of primary leukemia and lymphoma samples used in this study have been described previously (14) . In addition, 15 leukemia samples were obtained from the Cell Procurement and Banking Facility at The Johns Hopkins Oncology Center (Baltimore, MD) and 15 primary leukemia samples from patients presenting to the Pediatric Oncology service at Case Western Reserve University. The leukemia cell lines KG1A (AML), U937 (ALL), HEL (AML), ML-1 (AML), K562 (AML), HL-60 (AML), THP1 (AML), and RAJI (Burkitt’s lymphoma) were analyzed in this study. Cell lines were maintained in appropriate media and treated with 5 Aza-dC (Sigma) at a concentration of 1 µM for 3–5 days to achieve demethylation.

Genomic Cloning.
The CITB human bacterial artificial chromosome (BAC) library (Research Genetics, Huntsville, AL) was screened by PCR using p73 primers E2F: 5'-GCACCACGTTTGAGCACCTCTGG-3' and E3R: 5'-AGATGTAGTCATGCCCTCCAGGTG-3', and clone 190O18 was isolated. To isolate the 5' region of the p73 locus, an EcoRI sublibrary was generated from BAC 190O18 and was screened by colony hybridization using a 75-bp 5' terminal PstI fragment of the p73 cDNA (GenBank Accession number Y11416) as a probe. This screen yielded a 7.3-kb fragment encoding p73 exon 1. A map of the p73 5' region was generated by restriction digestion and by sequence analysis.

MSP.
Analysis of methylation patterns within the CpG island of the p73 gene in exon 1 (sequence -110-bp to -42-bp relative to translation start, GenBank Accession number Y11416) was determined after the chemical modification of genomic DNA with sodium bisulfite and MSP as described previously (15) . Primer sequences for p73 for the methylated reaction were 5'-GGACGTAGCGAAATCGGGGTTC-3' (sense) and 5'-ACCCCGAACATCGACGTCCG-3' (antisense), and for the unmethylated reaction were 5'-AGGGGATGTAGTGAAATTGGGGTTT-3' (sense) and 5'-ATCACAACCCCAAACATCAACATCCA-3' (antisense). Human placental DNA treated in vitro with SssI methyltransferase served as a positive control for the methylated reaction. Control reactions without DNA were performed with each PCR.

Southern Blot Analysis.
Genomic DNA (10 µg) was digested with methylation-dependent and methylation-independent restriction enzymes, separated in agarose gels, and transferred to nylon membranes as described previously (16) . Membranes were hybridized with ³²P-probes prepared by random hexamer primer extension, washed, and subjected to autoradiography.

p73 Transcript Analysis.
Cytoplasmic RNA was isolated and reverse-transcribed as described previously (17) . RT-PCR primers for p73 were 5'-CGGGACGGACGCCGATG-3'(sense, exon 1) and 5'-GAAGGTCGAAGTAGGTGCTGTCTGG-3' (antisense, exon 3). GAPDH expression was analyzed as described previously (18) .

SSCP Analysis.
Screening of the entire 13 coding exons (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) and intronic splice donor and acceptor regions of p73 was performed using primers described previously (19) . PCR reactions were carried out in a 10 µl-reaction volume containing 100 ng of genomic DNA and 1 µCi of [³²P]dCTP. Samples were diluted 1:10 in loading buffer (90% formamide, 1x Tris-borate EDTA, 10 mM NaOH, 0.05% bromphenol blue, and 0.05% xylene cyanol), heated at 90°C for 5 min, snap-frozen on dry ice, thawed on wet ice, and then loaded onto the gel (20) . Electrophoresis was performed using 1x MDE agarose (FMC BioProducts, Rockland, ME) with 5% glycerol in 0.5x TBE. Gels were run at 5–10 W at room temperature for 12–16 h, transferred to 3-mm Whatman paper, dried, and autoradiographed with Kodak X-OMAT film.

DNA Sequencing.
Aberrantly migrating bands as confirmed on at least two separate PCR/SSCP reactions were excised from the gel, submerged in 100 µl of dH₂O and heated to 80°C for 15 min. Ten µl of this elution containing single-stranded DNA was then reamplified using the appropriate primer set. Automated DNA sequence analysis was performed on PCR products directly or after cloning into pCR2.1-TOPO (Invitrogen).

	Results

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The p73 5' Region.
To facilitate analysis of methylation at the p73 locus, we isolated a human BAC clone by screening the library for an amplicon spanning intron 2. From this BAC, we isolated a plasmid clone containing p73 exon 1. Sequence analysis of this plasmid identified p73 exon 1 in a 0.5-kb EagI-NotI fragment (Fig. 1A) . Restriction mapping and further sequence analysis revealed the region surrounding exon 1 to be CpG- and GC-rich with abundant methylation-dependent restriction enzyme sites (Fig. 1A) , consistent with a CpG island (10) . An Alu repetitive element was identified in intron 1.

View larger version (50K): [in this window] [in a new window]   Fig. 1. A, a restriction map of the p73 5' region. A 7.3-kb EcoRI fragment containing p73 exon 1 was isolated and mapped by restriction enzyme digestion and by sequence analysis. , p73 exon 1; arrowhead, the orientation of transcription. , an Alu element in intron 1. Restriction endonuclease sites: E, EcoRI; P, PvuII; Ea, EagI; N, NotI; S, SacII; B, BamHI; above the map, the probe (pA5E.5) used in methylation analyses; *, location of MSP primers. B, Southern blot analysis of p73 methylation in leukemia cells. Genomic DNA was digested with EcoRI and NotI, methylation-sensitive enzymes. Samples were separated on a 1% gel and hybridized with the probe pA5E.5. A 7.3-kb band indicating methylation at the NotI site is clearly evident in lymphoid leukemia samples (Lanes 1–5 and 8) but not in myeloid leukemia samples. The very faint 7.3-kb band observed in three of the ALLs (Lanes 6, 7, and 9) represents minimal methylation; and these samples were not scored as positive.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Methylation of the p73 promoter region CpG island in cell lines and primary human samples. Visible PCR product in lanes marked U indicates the presence of unmethylated genes; visible PCR product in those lanes marked M indicates the presence of methylated genes. A, normal primary human tissues. All of the samples are unmethylated. B, MSP of p73 in human cancer cell lines. U937 (leukemia) is completely methylated, and HL60 (leukemia) is predominantly methylated. HS578T (breast) and SW48 (colon) contain both methylated and unmethylated p73. T47D (breast) and MCF7 (breast) were both unmethylated. C, MSP of p73 in primary ALLs and Burkitt’s lymphomas. All of the methylated primary tumors have evidence of unmethylated p73, a result of the presence of normal contaminating tissue. +, in vitro methylated DNA, is a positive control for methylation.

The methylation patterns we observed correlated with transcriptional silencing of the gene. For the fully methylated cell lines, KG1a and U937, p73 transcript was undetectable by RT-PCR (Fig. 3A) . Treatment with the demethylating agent 5 Aza-dC restored expression of p73 (Fig. 3A) , confirming the functional importance of methylation in epigenetic silencing of this gene. After several days, the treated cell lines demonstrated a slower growth rate and evidence of cell death. We could not attribute these observations solely to the restoration of p73 function, however, because 5 Aza-dC has a general toxic effect on cells which was independent of their methylation status at the p73 promoter (data not shown).

View larger version (63K): [in this window] [in a new window]   Fig. 3. Expression analysis of p73 by RT-PCR. The presence or absence of reverse transcriptase in RT reactions is indicated by + and -, respectively. Expression of GAPDH was performed on each sample to ensure the integrity of the RNA preparations. A, analysis of p73 expression in completely methylated leukemia cell lines, U937 and KG1A, before and after 5Aza-dC treatment. Note detectable product in both cell lines only after demethylation with 5Aza-dC. B, p73 expression in normal peripheral blood lymphocytes (NL) and bone marrow (BM). Each expresses p73. C, p73 expression in primary ALLs. By MSP, tumors 1–3 are methylated (M) and tumor 4 is unmethylated (U). The methylated tumors all show greatly reduced expression when compared with the unmethylated leukemia.

Southern blot analysis of methylation at the NotI site immediately 5' to exon 1 confirmed the above results and were concordant with the MSP analysis in 11 of 12 leukemia samples studied (Fig. 1B). In a group of nine ALLs, five were methylated by MSP, whereas six were methylated by Southern. The additional leukemia detected by Southern demonstrated a relatively faint 7.3-kb band indicative of methylation (Fig. 1B , Lane 8). Three AMLs were unmethylated by both techniques. In another group of leukemia samples analyzed only by Southern blot analysis, 22% (2 of 9) of ALLs were methylated, whereas all of the AMLs (n = 6) were unmethylated (data not shown). Thus, the incidence of p73 methylation that we observed in acute leukemias was comparable using the two different techniques.

The relatively high incidence of p73 methylation in ALLs and Burkitt’s lymphomas suggested that inactivation of p73 may be an important event in the etiology of these tumors. To further investigate the significance of this finding, we first studied whether methylation affected p73 expression in primary ALLs. p73 was fully expressed in normal lymphocytes and normal bone marrow (Fig. 3B) . In contrast, as was observed in the leukemia cell lines, methylation of primary ALLs correlated directly with transcriptional repression of p73 (Fig. 3C) . By RT-PCR, methylated ALLs demonstrated markedly lower levels of p73 transcript than an unmethylated ALL. Again, because some normal lymphocytes are invariably present in primary leukemia samples, this low-level expression in methylated tumors may reflect either transcription from unmethylated normal alleles or minimal expression from methylated tumor alleles.

While other tumor types have not been found to inactivate p73 by point mutation (4, 5, 6, 7, 8) , epigenetic inactivation of p73 in ALL suggested that ALL is a malignancy in which p73 mutations might occur. SSCP analysis was performed on 31 primary ALLs and 4 leukemia cell lines. We observed silent polymorphisms in exons 7 (Val245Val, GTGGTA), 9 (His349His, CATCAC), 14(ALA557ALA, GCGGCA), and 14 (ALA610ALA, GCGGCA) at frequencies of 6, 23, 11, and 17%, respectively (Fig. 4) . In addition, we detected a single base deletion in intron 3 and one polymorphism in intron 9 at frequencies of 17 and 14%, respectively. Thus, we found no mutations that caused an amino-acid substitution or frameshift.

View larger version (73K): [in this window] [in a new window]   Fig. 4. SCP Analysis of p73. Representative autoradiograph from exon 9. Lanes 1 and 2, primary ALL samples with the His349His, CATCAC polymorphism. Lanes 3 and 4, ALLs with wild-type sequence. Arrow, aberrantly migrating band.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

We initially determined that methylation changes within, and immediately flanking, exon 1 in the 5' CpG island of p73 do not serve as a primary mark for the imprinting of p73. Although we did not directly examine the allelic origin of p73 transcripts in this study, we did study those tissues that have been reported to demonstrate monoallelic expression consistent with imprinting, including normal lymphocytes, normal kidney, and neuroblastoma cell lines (1 , 9) . Each of these tissues and all of the normal tissue types that we analyzed were completely unmethylated. Although imprinting is often associated with methylation of CpG islands within the promoter of the imprinted gene, in some cases the relevant methylation changes occur at a distance. This is certainly true for the imprinted gene IGF-2, in which selective methylation of a second gene, H19, controls the paternal expression of IGF-2. Of note, H19 is located 75 kb distal to IGF-2 on chromosome 7 and, when silenced via methylation, allows enhancer elements to initiate IGF-2 transcription (21) . Thus, it remains possible that a methylated imprinting locus of p73 in normal tissues will be discovered outside the region we studied.

Importantly, we found that aberrant promoter methylation of p73 occurs frequently in ALL and Burkitt’s lymphoma. Promoter region methylation was tumor-specific since it was not observed in normal lymphocytes or bone marrow and resulted in markedly diminished expression of p73. Our results support previous observations that epigenetic silencing of tumor suppressor genes via methylation of promoter CpG islands is a common event in neoplasia. Indeed, in some cases, methylation changes are the predominant alterations that inactivate a tumor suppressor gene. For example, loss of expression of the DNA repair gene O⁶-methylguanine DNA methyltransferase in a variety of tumor types is caused primarily by promoter hypermethylation rather than deletion, mutation, or gene rearrangement (22) . Similarly, in sporadic endometrial carcinomas with microsatellite instability, defects in the DNA mismatch-repair gene MLH1 are more often due to hypermethylation than mutation (23) . Because we and others (4, 5, 6, 7, 8) have not detected any mutations of p73, aberrant methylation may be the only, or at least the most frequent, way that expression of this gene is altered in cancer.

Our results further suggest a tumor suppressor role for p73 in specific types of lymphoid hematological malignancies. Methylation changes were more common in, but not restricted to, T-cell tumors because B-cell ALLs and B-cell-derived Burkitt’s lymphoma were also involved. Loss of p73 could lead to defects in cell-cycle regulation and confer a selective growth advantage for clones in ALL and Burkitt’s lymphoma. Interestingly, although p53 abnormalities are the most common molecular lesions in human cancer (24) , they are relatively less frequent in de novo ALL (2–19%; Refs. 25 and 26 ) and non-HIV-related Burkitt’s lymphoma (30%; Ref. 27 ). Because p53 mutations are not common in these malignancies, one hypothesis is that loss of p73 represents an alternative event contributing to abnormal cell-cycle and cell-death regulation. The absence of p73 methylation in de novo AMLs, non-HIV NHLs, and CLLs and the fact that these tumor types also have a relatively low incidence of p53 mutations (28 , 29) imply different mechanisms of pathogenesis in these hematological malignancies.

	ACKNOWLEDGMENTS

 
We thank Drs. Fray Marshall (Emory University, Atlanta, GA) and Minoru Toyota (Johns Hopkins University, Baltimore, MD) for supplying tissue samples and James Voeller for technical advice regarding SSCP analysis.

	FOOTNOTES

 
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 This research was supported by NIH Grant CA43318 and by funds from the Oncology, Chemotherapy, Immunology, Biology Research Training Grant 5T32CA09071-19 (to P. G. C). J. G. H. and S. B. B. receive research funding and are entitled to royalties from ONCOR, which is developing products related to research described in this paper.

² These authors contributed equally to this article.

³ To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD, 21231. Phone: (410) 955-8506; Fax: (410) 614-9884; Email: hermanji{at}welchlink.welch.jhu.edu

⁴ The abbreviations used are: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; MSP, methylation-specific PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 5 Aza-dC, 5-aza-2'deoxycytidine; SSCP, single-strand conformational polymorphism; BAC, bacterial artificial chromosome; RT-PCR, reverse transcription-PCR; IGF-2, insulin-like growth factor 2.

Received 3/23/99. Accepted 6/ 1/99.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

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