Feb 3, 1984 - T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least ... established from an individual with hairy cell leukemia (7).
Vol. 4, No. 5
MOLECULAR AND CELLULAR BIOLOGY, May 1984, p. 890-897 0270-7306/84/050890-08$02.00/0 Copyright C) 1984, American Society for Microbiology
DNA Methylation and Expression of HLA-DROL MARVIN S. REITZ, JR.,'* DEAN L. MANN,2 MARIBETH EIDEN.l CECELIA D. TRAINOR,' AND MICHAEL F. CLARKE' National Cancer Instituite, Bethesda, Maryland 20205 Carcinogenesis,2 Laboratories of Tumor Cell Biology' and Hluman Received 26 October 1983/Accepted 3 February 1984
B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DRcx DNA sequences detected by a cloned DRc cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DRot mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DRa mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DRcx mRNA, and the partial demethylation of the DRcx DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DRo. mRNA; the HpaII sites within the BgIII fragment of DRo DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DRcx locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DRa locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.
HLA-DR antigens are cell surface proteins expressed by cells of lymphoid lineage and are important in T- and B-cell interactions (4). Most B-cells express DR antigens (2). Normal peripheral blood T-cells express very low levels of DR antigens(18). This expression is increased by mitogenic (23) or antigenic (5) activation in short-term culture. When peripheral blood T-cells from individuals infected with the human T-cell leukemia-lymphoma virus (HTLV) (25, 26, 28) (a retrovirus etiologically associated with certain adult T-cell malignancies [13, 30]) are put into short-term culture, they also express an elevated level of DR antigens compared with the homologous uncultured peripheral blood T-cells (D. L. Mann, unpublished data). T-cell lines established from HTLV-infected individuals or cord blood T-cells infected in vitro constitutively express DR antigens at high levels (6). In contrast, HSB and 8402, T-cell lines established from individuals with T-cell acute lymphocytic leukemia (T-ALL) (1, 24), do not express detectable DR antigens (24). We wished to determine the reasons for these differences in DR expression among different T-cell populations and have analyzed these cell lines for DRot and DRO mRNA as a measure of the transcriptional activity of these genes. We also analyzed DR(x at the DNA level. DNA methylation has been shown to be associated with the inactivation of a number of genes, including 3-globin genes (20), chicken ovalbumin genes (17), and proviral DNA of murine retroviruses (3, 8, 32). Consequently, we focused our attention on the methylation of DRot DNA sequences. The results of these experiments are presented below.
lished from an individual with T-ALL (24). CCRF-SB and HSB are similar B- and T-cell lines established from another individual with T-ALL (1, 24). CR-B and HUT-102 clone B2 are, respectively, an EBV-immortalized B-cell not infected with HTLV and an HTLV-positive T-cell line established from an individual with adult T-cell lymphoma who was infected with HTLV (6). MO cells are similarly T-cell line established from an individual with hairy cell leukemia (7) who was infected with HTLV Type 11 (14, 29). MJ (27) and CF-2 (D. L. Mann et al., J. Clin. Invest., in press) are DRpositive cell lines established from individuals with HTLVassociated T-cell neoplasms. Fresh peripheral blood T-cells were collected by lymphophoresis. T-cells were purified from normal lymphophoresis samples by nylon wool chromatography and were judged to be 95% pure by labeling with monoclonal antibody OKT-3. Samples obtained by lymphophoresis from individuals with adult T-cell leukemias were >95% OKT-3 positive and were used without purification on nylon wool. Cells were generally cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum. Normal T-cells (but not HTLV-infected T-cells) were activated with phytohemagglutinin for short-term cultures, and both cell types were grown in the continuous presence of Tcell growth factor as described previously (6). For some experiments, cells, were cultured in the presence of 10 or 20 ,ug of 5-azacytidine (Sigma Chemical Co., St. Louis, Mo.) per ml for 24 h. The medium was then changed, and the cells were cultured for another 72 h. For DR antigen expression assays, cells were labeled with the appropriate monoclonal antibody and analyzed by flow microfluorimetry with a FACS II microfluorimeter as described previously (18). Nucleic acid hybridization. High-molecular-weight DNA was purified and analyzed by Southern blotting essentially as previously described (33). Restriction endonuclease digestions were performed at 37°C for 16 h with 2 U of enzyme per p.g of DNA and the buffer recommended by the manufactur-
MATERIALS AND METHODS Cells. 8392 and 8402 are, respectively, an Epstein-Barr virus (EBV)-immortalized B-cell line and a T-cell line estab*
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TABLE 1. Surface antigens of T-cell lines Cellsa
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a.
% Positive cells' in monoclonal antibody: OKT3 OKT4 OKT8 3.1 3F10 37 13 3 1 96 3 7 21 2 97
8402 HSB 2 86 2 90 99 HUT-102 clone B2 2 49 MJ 18 68 91 a T-cell lines are described in the text. bCells were labeled with the indicated monoclonal antibody and analyzed by flow microfluorimetry as described in the text. 3.1 is a monoclonal antibody for HLA-DR antigens (generously provided by J. Strominger, Harvard University, Cambridge, Mass.), and 3F10 is a monoclonal antibody for HLA-(A, B, C) antigens (11). Numbers represent the percentage of positive cells after correction for background fluorescence.
HpaII and MspI were from New England BioLabs, Boston, Mass.; PvuII was from Boehringer-Mannheim Biochemicals, Indianapolis, Ind.; BglII was from Bethesda Research Laboratories, Gaithersburg, Md.; SmaI was from Promega BioTec, Madison, Wisc. Hybridization was performed for 16 h at 37°C in 2 ml of solution containing 0.45 M NaCl, 0.045 M sodium citrate (pH 7), 50% formamide, 5x Denhardt solution, and 10% dextran sulfate with a labeled probe (5 x 106 cpm). Filters were washed extensively at 65°C in 75 mM NaCI-7.5 mM sodium citrate (pH 7)-0.1 sodium dodecyl sulfate. Polyadenylic acid-containing RNA was purified by the guanidine hydrochloride method followed by oligodeoxythymidylic acid-cellulose chromatography and analyzed by Northern blotting after electrotransfer onto Gene Screen (New England Nuclear Corp., Boston, Mass.) as described previously (19). Cloned cDNAs representing mRNA for DRa (9) and DRPi (16) chains or for HLA class I mRNA (31) were labeled by nick translation with [32P]dATP and [32P]dCTP (800 Ci/mmol) (New England Nuclear) to a specific activity of 2 x 108 to 5 x 108 cpm/,ug. The DRa clone represents coding and 3' untranslated regions and encompasses ca. 1,050 bases of the DRa mRNA.
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RESULTS Correlation of DRa expression and DNA methylation in Tand B-cell lines. The cells used in these studies were tested with various monoclonal antibodies, including OKT-3, which is specific for certain subsets of T-cells, OKT-4, which detects an antigen on helper-inducer T-cells, and OKT-8, which detects an antigen on suppressor-cytotoxic Tcells. All of the EBV-immortalized B-cell lines used in this study express high levels of cell surface DR antigens. All of the tested HTLV-infected T-cell lines also express DR surface antigens (Table 1). The surface protein phenotype of HTLV-infected cells (Table 1) is typical of HTLV-infected leukemic blood T-cells (6) and differs from the surface protein phenotypes of the T-ALL T-cell lines 8402 and HSB, neither of which expresses detectable DR antigens. Polyadenylic acid-containing RNA from these cell lines was analyzed by Northern blotting to determine whether the lack of DR antigen expression correlated with the absence of DR mRNA. All of the B-cell lines, as well as the HTLV-infected T-cell line B2, expressed readily detectable mRNA of 1.6 to 1.7 kilobases (kb) for DRa and DR, (Fig. 1A and B, respectively). In contrast, the DR antigen-negative T-cell lines 8402 and HSB did not express detectable DR mRNA. (The bands of higher molecular weight result from the PBR322 moiety of the probe nonspecifically labeling the residual
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FIG. 1. Expression of DR mRNA in T- and B-cell lines. RNA purified from the indicated cell lines, subjected to gel electrophoresis on 1.1% agarose formaldehyde gels, and analyzed by Northern blotting as described in the text. RNA (3 ,ug) was used per lane. RNA was from cell lines 8392 (lane a), 8402 (lane b), SB (lane c), HSB (lane d), CR-B (lane e), or HUT-102 clone B2 (lane f). (A) Northern blots were hybridized to a DNA probe for DRax. (B) Northern blots were hybridized to a DNA probe for DR3. Arrows indicate the position of 18S and 28S rRNA markers. The gel was autoradiographed for 20 h at -70°C with a Quanta III high-speed was
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ribosomal precursor 18S and especially 28S rRNA, since 32P-PBR-322 alone labels these bands and since they are far more evident in blots with non-polyadenylic acid-containing RNA. This phenomenon has also been noted by others [22].) Digestion of human DNA with BglII yielded a fragment on Southern blots which contained all of the DNA sequences detectable with the DRcx probe (Fig. 2). This fragment was somewhat polymorphic from one individual to another and in the DNA samples tested here ranged from 4.8 to 4.0 kb. Cell lines 8392 and 8402 are heterozygous, containing two polymorphic alleles containing these sequences since BgIII digests of DNA from these cells contain both a 4.4- and a 4.0kb fragment. Digestion of the BglII fragment with MspI gave a fragment which varied from 2.8 to 3.4 kb (correlating with
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FIG. 2. Methylation of DRca DNA in T- and B-cell lines. DNA was purified from the indicated cell lines, digested with the indicated restriction endonucleases, and analyzed by Southern blots of 0.8% agarose gels with 20 to 25 i'g per lane as described in the text. (A) DNA was digested with BglII plus HpaII. DNA was from cell lines 8392 (lane a), 8402 (lane b). SB (lane c), HSB (lane d). and CR-B (lane e) and the HTLV-infected T-cell lines HUT-102 clone B2 (lane f), MJ (lane g), CF-2 (lane h), and MO (lane i). (B) DNA was digested with BgllI alone (lanes a, c, e, g, i. and k) or with Bglll plus Mspl (lanes b, d, f, h. j. and l). DNA was from cell lines 8402 (lanes a and b), HSB (lanes c and d), HUT-102 B2 (lanes e and f), MJ (lanes g and h), CF-2 (lanes i and j). and MO (lanes k and 1). Solid arrows indicate the positions of DRes fragments obtained by digestion with BgII alone, and open arrows indicate the positions of DRQ fragments obtained by digestion with BgII plus Mspl. Horizontal lines show the positions of HinidllI-digested k DNA fragments used as markers.
the relative size of the BglII fragment) and also contained all of the DRa sequences detected by the probe. Consequently, we analyzed the methylation of DRcx DNA sequences by using double digests with BgII and either of the isochizomers MspI (which cleaves DNA at CMeCGG) or HpaII (which will not cleave DNA at CMeCGG). The BgIII fragments from T-cell lines 8402 and HSB, which express no detectable DRot mRNA, were not affected by HpaII digestion (Fig. 2), indicating that all of the potential MspI sites are methylated. In DNA from all of the other tested cell lines (all of which express DR antigen and mRNA), some or all of the MspI sites are unmethylated, although generally some of these sites are methylated. These observations are supported by Southern blots of DNA with HpaII or MspI alone or with a double digestion with PvuII plus SmaI (which is also sensitive to methylation of cytidine) (data not shown). Although Southern blots with a DR,B probe are more complex and correspondingly more difficult to interpret than those with a DRot probe, digestion with HpaII and MspI showed that the ,B-related sequences were also more methylated in 8402 and HSB cells than in the other cells (data not shown). HSB and 8402 cells were cultured for 24 h in the presence of 5-azacytidine and assayed for DR expression 72 h later to determine wether DR expression could be induced. Azacytidine can be incorporated into DNA but not methylated, and subsequent rounds of DNA replication do not result in methylation of the newly synthesized DNA (12). Cells from both lines expressed readily detectable DR surface antigen
after but not before treatment with 5-azacytidine (Fig. 3A and B). DRcx mRNA became detectable in 8402 cells after growth in 5-azacytidine (Fig. 3C and Fig. 4B [lower center TABLE 2. Increase in DR antigen expression in peripheral blood T-cells after short-term tissue culture Cells' from patient:
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2 (12) ND' ND 32 (284) ND 0.5 (10) ND 16 (197) 97 (1.790) C 2 27 (68) 97 (1,763) C 33 (719) 48 96 (850) 2 11 (10) D 96 (946) 35 (464) D 48 96 (1,134) 13 (61) 2 E 95 (1,148) 20 (243) 48 E a Cells were peripheral blood T-cells collected as described in the text and cultured for the indicated time. Patients A and B are normal donors, and patients C, D, and E are HTLV-infected adult T-cell leukemia patients. b Cells were analyzed by flourescence-activated cell sorting as described previously (18) after being labeled with the appropriate monoclonal antibody as described in footnote b of Table 1. Numbers in parentheses are mean flourescence units, and the numbers not in are the percentages of positive cells. parentheses C ND, Not determined. A A B B
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and right-hand panelsl).The induction of DR mRNA synthesis and surface antigen expression correlated with a decrease in the degree of methylation of the DRa genes of these cells (Fig. 3D). Increased expression of DR after a short-term culture of Tcells. Uncultured T-cells from peripheral blood of both normal and HTLV-infected individuals expressed very low levels of DR antigens. The levels tended to be higher in the HTLV-positive cells (Table 2). In neither type of cells were all of the DRa sequences fully methylated, and there was no consistent difference between the two types of cells. After a short-term culture of these cells, DR antigen expression was greatly increased and again tended to be higher in HTLVinfected cells than in normal cells (Table 2). Dot blot and in situ molecular hybridization assays were performed on fresh peripheral blood cells from individuals with HTLV-associated neoplasms to determine whether the increase in surface antigen expression was correlated with an increase in DR
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RELATIVE FLUORESCENCE FIG. 3. Induction of DRcx in cells treated with 5-azacytidine. HSB and 8402 cells were treated with 10 and 20 ,ug of 5-azacytidine per ml, respectively (solid line), or were untreated (broken line), labeled with HLA-DR monoclonal antibody 3.1, and analyzed by flow microfluorimetry as described in the text. (A) HSB cells were analyzed. (B) 8402 cells were analyzed. (C) RNA was purified from 8402 cells treated with 5-azacytidine and analyzed by Northern blotting as described in the legend to Fig. 1 and in the text, except that 2 p.g of RNA was used per lane and autoradiography was carried out for 68 h. Arrows indicate the positions of 18S and 28S markers. (D) DNA was purified from 8402 cells treated with 5azacytidine and analyzed by Southern blotting after digestion with BgllI plus HpaI as described in the legend to Fig. 2 and in the text. Solid arrows show the positions of the DRat fragments after digestion with BglII alone, and the open arrows show the positions of the DRa after digestion with Bglll plus Mspl.
mRNA. DRa mRNA expression was detectable but relatively low (Fig. 4). After 48 h in culture, there did not appear to be a significant increase in autoradiographic grains in the in situ assay (Fig. 4A) or dot intensity in the dot blots (Fig. 4B) when the DRa probe was used, even though in one of these cultures DR expression was increased by more than 40-fold (Table 2). In contrast, HTLV mRNA increased four- to eightfold within this time period (Fig. 4B [lower right panel]; M. F. Clarke et al., Virology, in press). No changes were evident in the methylation levels of the DRa DNA sequences of these cells (Fig. 5). Similar results were obtained with DNA from peripheral blood T-cells from normal donors which are either not cultured or cultured for 7 days with phytohemagglutinin stimulation (data not shown). DISCUSSION EBV-immortalized B-cell lines and HTLV-infected T-cell lines express HLA-DR surface antigens, whereas T-ALL T-
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FIG. 4. DR mRNA expression in short-term tissue culture. (A) Cells were placed on microscope slides in a cytocentrifuge, fixed with ethanol-acetic acid (3:1), and hybridized in situ as described previously (15) with nick-translated 'H-labeled (5 x 106 to 10 x 106 cpm/,lg) cloned probes for HLA class I antigen mRNA (31) or a mixture of equal amounts of the DRa and DRI3 probes (9, 16). The slides were then dipped in photographic emulsion, autoradiographed for 3 weeks, and developed as described previously (15). HTLV-infected peripheral blood T-cells were cultured from patient C for 2 h (left-hand panels) or 48 h (right-hand panels) and hybridized with the mixed DR probes (top panels) or the HLA class I probe (bottom panels). The cells are visualized with Wright-Giemsa stain. Photomicrographs were taken at x40 magnification. (B) RNA was purified from 5 x 106 HTLV-infected peripheral blood T-cells as described in the text, except that the sodium acetate precipitation and chloroform-butanol extractions were omitted. The relative amount of RNA in each preparation was estimated by the amount of hybridization to dot blots with the probe for mRNA of class I antigens, the expression of which remained constant during the time period examined (Table 2). Based on this estimate, equal amounts of RNA were used for all of the cell types and serial twofold dilutions filtered onto nitrocellulose in a HybriDot manifold (Bethesda Research Laboratories, Gaithersburg. Md.) as described previously (Clarke et al., submitted for publication). The filters were then hybridized and autoradiographed as described in the text. The 32P probes used were inserts excised from clones of DRax (upper left and lower center panels), DR, (upper center panel), HLA class I antigens (pDPO-1) (upper right and lower right panels), or a probe for the CIaI-HindII fragment (CH-1) of the HTLV clone X CR-1 (19) (lower left panel). In the upper panels and the lower left panel, cells were cultured from patient D (lanes a and b) or E (lanes c and d) for 2 h (lanes a and c) or 48 h (lanes b and d). In the center and right lower panels, cell lines were MOLT-4 (a DR-negative T-cell line [lane al). 8402 (treated with azacytidine as described in the text [lane b]), and a highly DR-positive B-cell line from patient D (lane c).
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cell lines 8402 and HSB do not. We have shown that the lack of antigen expression is correlated with a lack of detectable DRa or DRf mRNA in these cells, suggesting that DR antigen expression in these cells is restricted at the level of transcription. The DRa DNA sequences in the cells tested which express DR antigens are all at least partially unmethylated at potential HpaII sites (although usually some methylation is evident). The presence of more than one band in HpaII-BglII digests of DRa-related sequences suggests either that there is a heterogeneity in some of these cultures in the degree of methylation of these sequences or that the two DRa alleles are methylated to a different extent. The DRa DNA sequences in the nonexpressing 8402 and HSB cells, in contrast, are completely methylated at the same sites. This suggests that there may be a correlation between methylation of a specific region of the DRa gene in these cells and the state of DRa mRNA transcription. Further support is lent to this hypothesis by the induction of DR expression in these cells after treatment with 5-azacytidine. Methylation has been shown to correlate with tran-
896
scriptional levels of 3-globin genes (20), chicken ovalbumin (17), and proviruses of some murine retroviruses (3, 8, 32), although there are also some genes in which methylation does not relate to low levels or lack of transcription (10, 21). Fresh peripheral blood T-cells from normal and HTLVinfected individuals express very low levels of DR antigen. A short-term culture results in an increase in expression. In spite of this increase, there is not a concomitant decrease in the level of methylation of the DRox DNA sequences or an increase in DRcx or DR3 mRNA. This suggests that in some situations, perhaps including T-cell activation, DR expression is regulated at other than transcriptional levels and is independent of DNA methylation. As judged by their cell surface markers, the T-ALL T-cells may be less mature than, and are at least different from, the HTLV-infected T-cells or peripheral blood T-cells. These data, therefore, suggest that DR expression is transcriptionally restricted in some types of T-cells by means which include methylation at certain sites in the DNA. In other Tcells, including those which are capable of an increase in the expression of DR antigens in a short-term culture, this does not appear to be the case. DNA methylation of DRot sequences in certain T-cells might thus correlate with a "coarse"' control of DR expression which is not operational in mature peripheral blood T-cells.
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ACKNOWLEDGMENTS We thank Dr. Larhammar for the DRc and DR, clones, S. Weissman for the HLA class I probe, F. Wong-Staal for HTLV clone pCH-1. J. Strominger for the 3.1 monoclonal antibody. A. Mazzuca for editorial assistance. and R. C. Gallo for advice and support.
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