T-Cell Receptor Chain Gene Rearrangement in Acute Myeloid Leukemia Always Occurs at the Allele That Contains the Undermethylated J/ l Region

The epigenetic phenomenon could play a role in the interaction between chromatin and DNA-binding enzymes, allowing us to consider an association between the phenomenon and gene rearrangement. The correlation between methylation status and rearrangement of the T-cell receptor (TCR) ~ chain gene in leukemia cells obtained from patients with acute myeloid leukemia (AML) was examined. All of the AML patients with a TCR-fl rearrangement had hypomethylated CCGG sequences within the J~l region on the rearranged allele, while the germline allele had completely methylated CCmeGG sequence in this region, indicating a strong association between hypomethylation status and rearrangement of the TCR ~ chain gene. In the DNA from AML patients with or without a TCR-~ rearrangement, the C/~2 region contained completely methylated CCmeGG sequences, even though they express T-cell-associated antigens, including CD7; this pattern is quite different from that observed in T-cell neoplasias. Moreover, some AML patients showed a TCR-~ rearrangement without the presence of immunoglobulin heavy-chain gene rearrangement, suggesting that TCR/~ chain gene involvement in AML is required for unknown factors other than common recombinase activity. I N T R O D U C T I O N Patterns of DNA methylat ion, i. e., the distribution of 5-mC as the fifth nucleotide in DNA, are cell type specific; thus this phenomenon may be impor tant in splicing genes and in cancer development (1, 2). Furthermore, methylat ion status of the calci tonin gene has been shown to correlate with the progression of chronic myelogenous leukemia (3, 4). These data indicate that epigenetic changes may play a role in genetic alterations in cancer cells (5). It has been demonstrated that methylat ion status in the TCR 4 ~ chain gene is different in different types of hematopoiet ic cells (6-8); for example, TCR ~ chain genes in normal mature B-cells are highly methylated, whereas those of mature and neoplastic T-cells are not (6, 7). We have shown a close relat ionship between hypomethyla t ion status and rearrangement of the TCR/~ chain gene in B-precursor ALL; the allele that contains a TCR/3 chain gene rearrangement always carries hypomethylated loci within the C~I region (8). The current study aims to determine the association between hypomethyla t ion pattern and rearrangement of the TCR/3 chain gene in A M L cells. It is currently known that a cross-lineage recombinat ional event may be due to a putative common recombinase activity (9); however, some A M L patients had a Received 5/21/92; accepted 9/18/92. 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. Supported in part by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science, and Culture (04671536). 2 To whom requests for reprints should be addressed, at First Department of Internal Medicine, Tokyo Medical College, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan. 3 Present address: Walther Oncology Center, Indianapolis, IN 46202-5121. 4 The abbreviations used are: TCR, T-cell receptor; ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; IgH, immunoglobulin heavy chain; IgJn, a joining region of the immunoglobulin heavy chain; V(D)J, variable (diversity) joining; RAG, recombination activating genes. TCR-~ rearrangement without a rearranged IgH chain gene (10-15). Therefore, in such cases of AML, it seems difficult to hypothesize a common recombinase activity that produces IgH and TCR rearrangement. This study provides further insight into the recombinat ion of the TCR ~ chain gene in human nonlymphoid neoplasias, based on the epigenetic phenomenon. M A T E R I A L S AND M E T H O D S Patient Samples. To identify the IgH and TCR-fl rearrangements, we first examined 101 patients with AML using IgJH and TCR-~ probes (see "Gene Probes" below). The diagnosis of AML was based on the French-American-British criteria. After informed consent was given by the patients, enriched leukemia cells (>85% blasts) were obtained by the Ficoll-Hypaque density gradient method from heparinized peripheral blood or bone marrow aspirates. As a control, granulocytes were obtained from hematologically normal individuals. DNA Preparations and Southern Blot Analysis. High-molecularweight DNA was extracted from mononucleated cells of each patient by a urea lysis/cesium chloride method (16). Ten #g of DNA were digested with the appropriate restriction enzymes (Takara Shuzo, Kyoto, Japan), according to the supplier's recommendations. Digested DNA was size-fractionated by 0.8% horizontal agarose gel electrophoresis. The gel was denatured in 0.25 U NaOH and 0.6 M NaCI and neutralized in 0.6 m NaCI and 0.24 M Tris-HCl. The DNA fragments were transferred to a nylon filter (Zetabind; AMF, Cuno, CT) and cross-linked by exposure of shortwave UV light for 5 min. The filter was prehybridized and then hybridized with randomly primed 32p-labeled DNA probe. After the hybridization, the filter was washed, air dried, and exposed to Fuji XR film for various periods using an intensifying screen (16). Gene Probes. Immunoglobulin heavy chain gene rearrangements were detected using 3-kilobase EcoRI/HindIII-digested IgJH as a probe (17). A 0.72-kilobase A vaI/EcoRI-digested C/~ 1 probe, isolated from the HBVT 96 clone, was used to detect rearrangement of the TCR/~ chain gene (18). This probe hybridizes with 12and 4-kilobase EcoRIdigested germline fragments that contain C/~I and C/~2, respectively. The 0.77-kilobase PstI-digested Jur~2 probe detects 4(corresponding to C~2) and 5-kilobase (J~2) germline fragments (19). To identify the TCR 6 chain gene rearrangement, a 1.5-kilobase EcoRI germline fragment containing the ajoining region of the human TCR J61 chain gene and a 1.5-kilobase EcoRI germline fragment containing the constant region of the human TCR 6 chain gene (C~I) were used (15). Analysis of IgJH was performed with DNA digested with HindIII, EcoRI, or BamHI. EcoRI-, BamHI-, and HindIII-digested DNAs were separately evaluated with the C~I probe, and EcoRI was utilized for the Jur/~2 probe. J61 analysis utilized EcoRI and BamHI, and C61 utilized EcoRI-digested DNA. Methylation Sites of the TCR ~ Chain Gene. To assess methylation status, leukemia cells obtained from 30 AML patients, including 7 patients with CD7 + AML, were examined (Table 1). The DNA was digested overnight at 37~ with 20 units of EcoRI and HpaII; 20 units of HpaII were added for another overnight period (double HpalI digestion) to avoid incomplete digestion (8). The methylation sites of the TCR ~ chain are scattered from MI to M30 (Fig. 1) (6-8). When CCGG sites in the M1-M7 region are unmethylated, the fractionated size of DNA digested by EcoRI plus HpaII reduces to less than 12 kilobases; on the other hand, when this region has a completely methylated CCmeGG sequence, the fractionated size