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Subsets of hairy cell leukemia defined by unique membrane proteins
Leukemia Research. Vol. 4. No. 5, pp. 477--488 © Pergamon Press Ltd.. 1980. Printed in Great Britain.
SUBSETS
0145-2126/80/1001~)477102.00/O
OF HAIRY CELL LEUKEMIA UNIQUE
MEMBRANE
DEFINED
BY
PROTEINS
ROBERT C. SPIRO, MOTOHIKO AIBA, ISAO KATAYAMA,* PHILIP P. RAFFA, KIYOSHI SAKAMOTO, DAVID T. PURTILO, JOHN L. SULLIVAN and ROBERT E. HUMPHREYS Departments of Pharmacology, Pathology, Medicine and Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester, MA 01605, U.S.A. (Received 15 May 1980. Accepted 11 June 1980)
Abstract--Leukemic cell membrane proteins of 27 patients with hairy cell leukemia were evaluated by SDS gel eleetrophoresis in order to identify subsets of these patients and to define molecules for isolation and the development of diagnostic reagents. [JSS]methionine metabolic labeling, gel electrophoresis of isolated membranes and fluorography demonstrated about 100 membrane proteins. Variations in the amount of synthesis of two principal proteins, p35 and pl 5 (35,000 and 15,000 dalton proteins respectively),defined, in relative terms, thrc¢ subtypes of hairy cell leukemias: (a) p35*, p15- cells, (b) p35-, p15 ÷ cells, and (c) p35-, p15- cells. At this time, it is not known whether these subsets represent various related disease entities or stages in the progression of one disorder. Although p35 migrated in the gels with one component of the HLA-DR (p23, 30) protein complex, immunoprecipitation studies with anti-p23, 30 serum indicated that it probably was not derived from HLA-DR antigen, which was, in facL synthesized in addition to p35 on all hairy cells which were tested. Cultured lymphoblastoid cell lines established from the peripheral blood of hairy cell leukemia patients were substantially different m membrane protein patterns from the fresh leukemic cells. In particular, p35 and p15 were minimally expressed by cultured cells.
INTRODUCTION VARIATION in survival times among patients with one morphologically defined leukemia has been ascribed to either (a) different degrees of host immune response to control the growth of the malignancy, or (b) the existence of as yet unrecognized leukemia subsets which differ in biological aggressiveness and, thus, in clinical course. The role of the immune system, and particularly of suppressor T lymphocytes, in regulating tumor growth is becoming defined [23]. In addition, cell surface markers have been used to identify biological subsets of leukemias, in some instances with distinct clinical courses and requirements for therapy, e.g. the definition of the p23,30-positive subset of acute lymphoblastic leukemia [28, 30]. With a view towards examining lymphoid malignancies for patterns of membrane proteins which might define subsets, we have refined a technique for examining [JSS]methionine-labeled, leukemic cell membrane proteins by clectrophoresis in SDS polyacrylamide gradient gels [27]. With this method over 100 membrane proteins can be identified. Some proteins are uniquely expressed on T or B cell lines, or at stages of activation of normal lymphocytes ([33], and W. Boto and R. E. Humphreys, unpublished observations). Externally exposed proteins can be defined by su.sceptibility to lactoperoxidase-mediated radioiodination, fucose labeling, or papain cleavage [26]. HLA-A, -B and HLA-DR antigens can be identified by coelectrophoresis *Current address: Department of Pathology. Saitama Medical School. Moro Machi. Saitama, Japan. Abbreviations: EBV. Epstein-Barr virus: HCL. hairy cell leukemia; SDS, sodium dodecyl sulfate. 477
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of immunoprecipitated antigens which are recognized witl~ heteroantisera to p44, 12 and p23, 30, respectively [27, 33]. In applying this method of analysis to lymphoid tumor cell lines, we found a close similarity in membrane protein patterns between a group of Burkitt's lymphoma cell lines and a series of EBV-transformed cells from normal individuals, indicating that in vivo and in vitro transformation with EBV yielded cells frozen at one stage of differentiation [33]. Having established the reproducibility, validity and potential diagnostic value of this method of analysis, we have directed our studies to the leukemic cells of a group of 27 patients with hairy cell leukemia (HCL; leukemic reticuloendotheliosis). The objective of these studies was to describe the membrane protein patterns of freshly obtained leukemic cells in order to identify subsets of patients and to identify unique proteins which might be isolated for development of immunodiagnostic reagents. In addition, cultured cells lines, which were established after EBV infection of the peripheral blood of HCL patients, were examined in order to assess the differences between fresh malignant cells and immortalized cells which were cultured for some time in the absence of hormonal or immunoregulatory influences of the host. MATERIALS AND METHODS Leukemic cells For metabolic labeling with l'3SS]methionine, mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation from the peripheral blood of 27 patients with HCL and one patient (No. l) who presented as a patient with HCL until examination of his spleen, in the course of this study, revealed a well differentiated lymphocytic lymphoma with plasmacytoid features. For all other patients, the diagnosis of HCL was made on the basis of characteristic clinical, hematologic, and pathologic features which included the following: (1) the demonstration of cell surface microvilfi and pseudopods in all 27 cases 1"21,(2) the finding of the ribosome-lamella complex in 14 of 27 patients [15], (3) the presence of positive tartrate-resistant acid phosphatase reactions in 25 of 27 patients [12"l, (4) bone marrow biopsies showing a loose mononuclear cell infiltration with intercellular reticulin fibrosis in 15 of 15 patients from whom such biopsies were obtained [31 and (5) the splenic histopathoiogy demonstrating diffuse mononuclear cell infiltration of the red pulp with formation of pseudosinuses and atropy of the white pulp in 20 of 20 patients [14, 221. In addition, peripheral blood samples from each patient were reevaluated at the time of this study by phase contrast microscopy (demonstrating some to many pathognomonic hairy cells from each patient), electron microscopy and enzyme liistochemistry of tartrate-resistant acid phosphatase and a-naphthyl butyrate esterase activity [91. These examinations were conducted in accordance with the diagnostic principles of multiple investigators [2.3,9. 12, 14, 15, 221. EB V-immortalized cell lines Lymphoblastoid cell lines were established after exposure of Ficoll-Hypaque isolated cells to supernatant suspensions of the B95-8 strain of Epstein-Barr virus (EBV) [20]. Immunofluorescence analysis of these cultured cell lines with reagents specific for IgM, IgD, kappa and lambda determinants were performed [36]. Radiolabeling of cells and SDS electrophoresis of membranes and immunoprecipitated proteins Previously described techniques were followed for labeling cells with [3SS]methionine, isolation and electrophoresis of membranes, preparation of Staphylococcus aureus-bound immunoprecipitates with heteroantisera, and electrophoresis of these precipitates and [14CJacetylated proteins as molecular weight standards [4.16,17,19,26.27]. The preparation and characterization of anti-p44.12 (HLA-A, B antigen and //2-microglobulin) and anti-p23,30 (HLA-DR antigen) sera have been reported [5, I01.
RESULTS Membrane proteins of leukemic cells Variable membrane protein patterns were observed among the ~35S]methioninelabeled, Ficoll-Hypaque-separated, peripheral blood, lymphoid cells of the HCL patients (Fig. l). The membrane protein patterns of the mononucl~r cell preparations reflected principally incorporation into leukemic cells because, in separate experiments, leukemic cells and cultured lymphoblastoid cells were found to incorporate [35SJmethionine at
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FIG. 1, Membrane proteins of hairy cell leukemias, r3~S'imethionine-labclcd, detergent-solubilized membranes of Ficoll-Hypaque-separated lymphoblasts of HCL patients were subjected to electrophoresis in polyacrylamide gradient slab gels. Three subsets of patients could be identified on the basis of relative degrees of synthesis of 35,000 and 15.000 dalton proteins: (a) p35 ÷, p15-, (b) p35-, p15 *, (c) p35-, p15- groups. [J4C']acetylated proteins served as molecular weight standards. HSB and SB are T and B cell lines from one patient (21).
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FIG. 3. Coelectrophoresis of immunopreeipitated p44, 12 (HLA-A, B antigen and /},-microglobulin) and p23, 30 (HLA-DR antigen) with HCL membrane proteins. In order to identify the relationship of HCL membrane proteins to HLA-A, -B and HLA-DR antigens, immunopurified HLA antigens were subjected to electrophoresis. Although p35 ran at the same Rf value as did one component of the HLA-DR complex of 3 proteins (arrows), it was judged not to be derived from HLA-DR antigen for several reasons which are discussed in the text. 481
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F[o. 4. Comparison of freshly obtained leukemic cells with cultured lymphoblasts of the same patients. Membrane proteins of freshly obtained leukemic cells (F) and of cultured lymphoblastold cell lines (C)of individual patients were compared, p35 and p15 were hardly detected on the cultured line and many other differences in expression of minor proteins were found. 482
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rates ranging from 8- to 20-fold the rate of incorporation by normal peripheral blood lymphocytes [33]. The differences in protein band intensities among the various" HCLs fell into two broad categories: (a) variations in levels of synthesis of p35 and p15 (two proteins of 35,000 and 15,000 daltons, respectively) which were prominently synthesized in cells of only some patients, and (b) variations in synthesis of less prominent, or relatively minor, membrane proteins. Four patients (cases 2, 3, 4 and 8) had very high levels of synthesis of p35 (p35 +, p15- type of HCL). Four other patients (cases 1, 9, 13 and 14) had high levels of synthesis of p15 (p35-, p15 + type of HCL). A third group of patients (cases 10, 11 and 12) had low levels of synthesis of p35 and minimal or no apparent synthesis of pl5 (p35-, p15- type of HCL). It should be noted that characterization of these subsets on the basis of p35 synthesis was relative, for although very great amounts of p35 were synthesized by the first subset of HCL, some p35 was in fact observed in membranes of the other two subsets of HCL. Furthermore, a second refinement in nomenclature might be made. That is, "pl 5" constituted 3 proteins about 14,000 15,000 and 16,000 daltons, with only the most prominent one giving its name to this group, In this study, although 28 patients were examined by this methodology, completely satisfactory membrane gels were obtained with calls from only 11 patients. The other 17 patients with low peripheral blood white cell counts (less than 2000 cells/mm 3) did not yield high enough levels of [3SS]methionine incorporation to permit technically adequate gel electrophoresis of their membrane proteins. Consequently, the frequency of patients which were (a) p35 +, p15-, (b) p35-, p15 + and (c) p35-, p15- could not be assessed reliably. There was no correlation between percentages of leukemic cells and white cell counts on the one hand and membrane protein patterns on the other. For example, the white cell counts and percentages of leukemic cells of selected patients were as follows: patient 2, 28.8 x 103WBC, 93% LC (leukemic cells); patient 3, 13.7 x 103WBC, 72Y/o LC; patient 10, 2.3 x 103WBC, 19% LC; patient I, 23.5 x 103 WBC, 41Yo LC; patient 9, 6.9 x 103 WBC, 10% LC. The proteins which were defined in these gels were judged to have been synthesized 90-99% by the leukemic hairy cells because, although normal mononuclear cells were also isolated by the Ficoll-Hypaque gradient, we have shown in previous studies that normal, peripheral blood lymphocytes under these conditions of incorporation, synthesize 1,3SS]methionine into proteins at 5-8% of the rate of leukemic cells i5, 33]. The amounts of the 1,3SS]methionine incorporated into proteins of normal mononuclear cells would be negligible in most instances of this study. Furthermore, in the case of p35-positive patient 2, for example, 93% of the cells were leukemic hairy cells and in pl5-positive patient 1, 41% of the cells were leukemic cells, p35 and p15 thus were clearly synthesized by the leukemic cells of those respective patients. Differences in expression of many minor membrane protein bands were found among the HCL patients but these variations did not form dominant patterns. For example, patient 2 well synthesized a 75,000 dalton protein which was not readily apparent among other patients' cells. Considerable heterogeneity among membrane proteins in the 43,000-48,000 dalton weight range was noted among cells of all patients. Proteins about 28,000 daltons were more extensively synthesized by the four patients' cells which were p35 +, p 15-. Other bands, especially in the 80,000-150,000 dalton range, appeared in cells of a few patients, but their expression did not correlate with the apparent subsets of HCL so clearly defined by levels of synthesis of p35 and p15. As controls for these experiments, the membrane proteins of HSB (from a patient with T cell ALL) and SB (the B lymphoblastoid line from the same patient) I-l], were subjected to electrophoresis in parallel to the HCL samples. Bands which were identified in separate experiments to be HLA-DR antigen (by recognition with anti-p23,30 serum) were present on SB but not on HSB. T cell-related proteins of 18,000, 35,000 and 110,000
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ROBERT C. SplltO et al.
daltons were identified on HSB. [t4CJacetylated proteins were used as molecular weight standards, but the molecular weights of membrane proteins in the Laemmli gel system can only be considered as approximations for reference purposes [271. The function of these many proteins were unknown. However, since p35 migrated at the position of HLA-DR antigen, its relationship to human Ia-like proteins was tested in immunoprecipitation experiments.
Immunoprecipitation of HLA antigens from hairy cell leukemia lymphoblasts lmmunoprecipitates of nonionic detergent-solubilized membrane proteins and heteroantisera to p44,12 (HLA-A, -B antigens and fl2-microglobulin) and to p23,30 (HLA-DR antigen) were obtained with formalinized Staphylococcus aureus. These immunopurified HLA molecules were subjected to electrophoresis in a 147/o polyacrylamide slab gel (Fig. 2). HLA-A, B antigens and fl2-microglobulin were constantly expressed on all HCLs tested by this method. Likewise, HLA-DR antigens were precipitated from each HCL, but a variability in structure among the patients was found. We, and others, have also observed this heterogeneity in apparent molecular weight of HLA-DR components among lymphoblastoid cell lines [:11, 33]. This variability in pattern could reflect structural differences among alleles at HLA-DR loci, as is reported for the I-A and I-E/C loci of the mouse [13, 35]. In order to characterize the relationship of HLA-DR antigen to p35 which was heavily synthesized by HCLs, immunopurified HLA molecules were subjected to electrophoresis along with samples of membrane in a SDS polyacrylamide gradient gel (Fig. 3). HLA-A, -B antigen and flz-microglobulin were identified both in the precipitates and among the membrane proteins. The HLA-DR antigen was resolved into three bands at apparent molecular weights of 32,000, 35,000 and 37,000 daltons, which have been described previously [20]. p35, the extensively synthesized protein on some HCL cells, comigrated with the 35,000 dalton component of the HLA-DR antigen. However, p35 was thought not to be derived from HLA-DR antigen, because a large amount of p35 was not precipitated with anti-p23,30 serum and because the structure of the HLA-DR antigen which was obtained from cells expressing p35 was identical to that from HCL cells not expressing p35 (and to HLA-DR from B lymphoblastoid cell lines, e.g. RAJI, SB, LAZ-007) [5, 27, 33]. If the p35 on some leukemic cells had been part of the HLA-DR complex, then precipitation with anti-p23,30 serum might have recognized more distinctly the unique 35,000 dalton protein which was so heavily synthesized. Further confirmation of the structural difference between p35 of the p35 +, plS= type of HCL and HLA-DR antigen can be obtained through additional chemical studies. Differences in membrane protein patterns between freshly obtained leukemic cells and cultured lymphoblastoid cell linesfrom individual patients The objective of this experiment was to assess whether the membrane protein patterns of freshly-obtained HCL tumor cells were similar to the membrane protein patterns of EBV-transformed, cultured lymphoblastoid cell lines which were established from the peripheral blood of the respective HCL patients. For five patients from whom lymphoblastoid cell lines were established, membrane proteins of cultured lines (C) were subjected to ¢lectrophoresis in wells adjacent to membrane proteins of the respective freshlyobtained HCL tumor cells (F) (Fig. 4). Membrane protein patterns of the EBV-transformed lines from five patients resembled each other and resembled lymphoblastoid cell lines established from normal individuals and from Burkitt lymphomas [33]. The cultured lymphoblastoid cell lines were distinctly different from freshly obtained leukemias. p35 and pl20 disappeared or were greatly reduced in the established cell lines. For patients l and 14, the pl5 protein was absent or was minimally present in the cultured
Membrane proteins of HCL
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cell lines. In addition, differences between cultured cell lines and fresh tumors were seen in many other membrane proteins. For example, p40 and IM6 were more prominently synthesized by cultured lines than by the fresh leukemias. While many of the minor membrane proteins appeared to be the same in the fresh leukemic cells and in the cultured lymphoblastoid cells, the dramatic differences in the expression of several heavily synthesized proteins underscored the finding that fresh ieukemias and the cultured lines were distinctly different. In particular, p35 and plS, which were diagnostic for apparent subsets of hairy cell leukemia, were not well synthesized or were absent from the cultured lymphoblastoid cell lines which were established from patients whose leukemic cells had expressed these respective proteins. Because the five cultured lymphoblastold cell lines were polyclonal for expression of surface IgM and surface IgD and kappa and lambda light chains, they were considered to be derived by EBV-transformation of normal B lymphocytes and not from the malignant leukemic cells.
DISCUSSION The goal of this study was to compare the membrane proteins of tumor cells from a series of patients with hairy cell leukemia (HCL). With a finely resolving methodology of [3~S]methionine labeling and polyacrylamide gel electrophoresis, we have identified membrane protein differences which define three phenotypes of HCL patients. Two heavily synthesized proteins were identified as candidates for isolation and the preparation of immunodiagnostic reagents. In addition, substantial differences in membrane protein patterns were found between fresh leukemic cells and cultured cell lines established from the respective patients. Each of these points can be considered in detail. The membrane protein patterns of different HCL patients varied in their expression of two dominantly synthesized proteins: p35 and plS, proteins of 35,000 and 15,000 daltons, respectively. Three types of cell membrane protein patterns could be identified in relative terms: (a) p35 ÷, p l S - leukemias, (b) p35-, p15 + leukvmias, and (c) p35-, p l S - leukerajas. A close examination of Fig. 1 shows 35,000 dalton protein(s) on all HCL's, although in relative terms, one subtype expressed large amounts (p35 +) and two other types expressed reduced amounts (p35-). The 35,000 dalton proteins on the latter types of cells might not be the same p35 protein which was synthesized by the first subtype of HCL. For example, it could be part of the "underlying" HLA-DR complex. In contrast, p15 + cells showed large amounts of 15,000 dalton protein(s)and p15- cells appeared to synthesize no pl 5. The absolute positivity or negativity of these subsets of leukemias for p35 and p15 can only be established when diagnostic antisera have been prepared to the purified proteins. Such antisera will simplify analyses for these subsets of HCL and will permit screening of other lymphoid malignancies for the presence of p35 and p15. For example, at least one patient with chronic lymphocytic leukemia expressed a 15,000 dalton protein which might cross-react serologically with the p15 found on some HCL patients' cells [32]. Furthermore, patient 1 in this study, who heavily synthesized plS, had a diffuse lymphocytic lymphoma with plasmacytoid features masquerading as hairy cell leukemia. p35 was thought not to be a component of the HLA-DR (Ia antigen-like) complex because (a) large amounts of p35 were not precipitated with anti-p23,30 serum and (b) the HLA-DR proteins which were precipitated from cells which heavily expressed p35, were very similar to the HLA-DR molecules expressed on other cell lines such as B lymphoblastoid cell lines from normal individuals and Burkitt lymphoma cell lines. In addition to observing that p35 is apparently unrelated to HLA-DR antigen, we have, in fact, seen heavy synthesis of several novel 35,000 dalton proteins by some other cells. For example,
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48-h concanavalin A-activated, E rosette-purified T lymphocytes and 48-h P3HR-I virus superinfected RAJI cells both synthesized relatively large amounts of approximately 35,000 dalton proteins ([33]; W. Boto, D. Casareale, and R. E. Humphreys, unpublished observations). None of these proteins were recognized with anti-p23,30 serum in immunoprecipitation assays. Their relationship to p35 which is seen on HCL cells is unknown at this time, other than their coincidence in size. As with p35, the identity and function of p15 on HCL is unknown, p15 migrated at an R~ value equal to the [:~4C'Jacetylated lysozyme molecular weight standard. However, it probably is not lysozyme because (a) immunoperoxidase studies have failed to find lysozyme on HCL cells in general [:24], (b) patient 1 who had abundant plS on his leukemic cells, had negligible or absent urinary and serum lysozyme, and (c) a rabbit anti-human lysozyme serum (Bio-Rad Labs; catalog No. 170-1572) failed to immunopreo cipitate p15 under conditions at which anti-p44,12 and anti-p23,30 sera recognized their respective antigens. It is not clear at this time whether the phenotypically distinct subsets of HCL, as we have described them here, represent different forms of related diseases or different stages in the progression of one disorder. A cursory review of pathological and clinical findings in these patients does not indicate correlations to the stage, severity, or prognosis of their disease. However, after a year we plan to repeat this study in order to assess possible transitions in phenotype patterns of these patients and to extend our investigation in a systematic fashion to correlations with clinical and pathological findings. The observation that cultured lymphoblastoid lines established from the peripheral blood of patients with HCL failed to express membrane protein patterns of the freshlyobtained malignancies has adverse implications for the preparation of diagnostic reagents. Because the lymphoblastoid lines were polyclonal with respect to expression of surface immunoglobulin, they did not represent the original malignant cell but were derived from EBV-transformed, normal B lymphocytes r36]. Their membrane protein patterns were dramatically different from those of the fresh tumors. In particular, two principal proteins of the tumors, p35 and plS, appeared to be absent from the lymphoblastoid cell lines. Therefore, the cell source for the future isolation of p35 and p15 can only be the fresh leukemic cells. In surveys of fresh leukemias and activated normal T lymphocytes, we have found numerous examples of molecules which are not expressed or are minimally expressed on cultured iymphoblastoid cell lines (I-32, 33]; W. Boto, J. U DeMartino, R. C. Spiro, and R. E. Humphreys, unpublished observations). Consequently, for the isolation of proteins to generate immunodiagnostic reagents, one is forced to turn to fresh leukemic cells or to cultures of activated normal lymphocytes for their respective proteins. One cannot, in fact, take advantage of the large volumes of uniform cells which can be raised with cultured lymphoblastoid lines. Finally, a point of caution is implied in our observation that leukemic cells are different from the EBV-transformed cultured cell line established from the same patient's blood. That is, in the case of other examples of "tumor cell lines," both with respect to HCL [6, 7, 8, 18, 21, 25, 29, 31, 34] and to other tumors, the cultured cells might be quite different from the fresh tumor when membrane protein patterns are analysed by this method. In summary, we have refined a method to examine over 100 membrane proteins of lymphoid malignancies and cell lines, employing r3SS]methionine metabolic labeling and high resolution SDS polyacrylamide gradient gel electrophoresis. Differences were described in membrane proteins among patients with HCL. Three phenotypically different types of patterns were observed on the basis of levels of synthesis of p35 and plS. Although p35 migrated at the weight range of HLA-DR antigen, it was shown to be distinct serologically from that antigen complex. In future studies, we will assess the potential for progression of these subtype patterns of membrane proteins during the
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course of the disease and whether these subtype patterns do, in fact, correlate with clinical and pathological findings and have prognostic implications. Acknowledgements--This study was supported by NCI contract CB 64072 and NCI grant CA 25873 (to R.E.H.), by ACS grant P D T - I M (to I.K.), by NCI grant CA 23561 (to D.T.P.), and by ACS grant IN-129 (to J.L.S.). R.E.H. was the recipient of a Cancer Research Scholar Award from the American Cancer Society, Massachusetts Division, Inc. The expert secretarial assistance of Ms. Lena DeSantis in the preparation of this manuscript is gratefull) acknowledged. We very deeply appreciate the collaboration of many physicians for referring to us their patients, for discussing this project with us and with their patients, and for giving advice and comments.
REFERENCES 1. ADAMSR. A., FLOWERS A. & DAVIS B. J. (1968) Direct implantation and serial transplantation of human acute iymphoblastic leukemia in hamsters. SB-2. Cancer Res. 28, 1121-1125. 2. BOURONCLEB. A. (1979) Leukemic reticuloendotheliosis (hairy cell leukemia). Blood 53, 413-436. 3. BURKE J. A. (1978) The value of the bone-marrow biopsy in the diagnosis of hairy cell leukemia. Am. d. Clin. Path. 70, 876-884. 4. CRAWFORD L. V. ~' GESTELAND R. F. (1978) Synthesis of polyoma proteins in vitro, d. Mol. Biol. 73, 627-634. 5. FALDETTA T. J,, HOWE R. C., ROGAN K. M., SPIRO R. C., KATAYAMAI., PECHET L. & HUMPHREYS R. E. (1978) Demonstration of the internal synthesis of p23,30 by several lymphoid malignancies. Exp. Hemat. 7, 94-104. 6. GOLDE D. W., QUAN S. G. & CLINE M. J. (1976) Hairy cell leukemia. In vitro culture studies. Ann. Intern. Med. 85, 78-79. 7~ GOLD[/ D. W., STEVENS R. H., QUAN S. G. & SAXON A. (1977) Immunoglobulin synthesis in hairy cell leukaemia. Br. J. Haemat. 35, 359-365. 8, HEYDEN H. W., WALLER H. D., PAPE G. R., BEN6HR H. CBR., BRAUN H. J., WILM$ K., RIEBER E. P. & RIETHM/JLLER G. (1976) HaarzelI-Leukiimie I. Klinik, Zytocbemie, Phagnzytosef~ihigkeit yon Haarzellen, Etablierung permanent washsender Zellinien. Dtsch Med. Wochenschr. 101, 9. 9. HIGGY K. E., BURNS G. F. & HAYHOE F. G. J. (1978) Identification of hairy cells of leukemic reticuloendotbeliosis by an esterase method. Br. d. Haemat. 38, 99-106. 10. HUMPHREYS R. E., McCUNE J. M., CHESS L., HERRMANN H. C., MALENKA D. J., MANN D. L., PARHAM P., SCHLOSSMAN S. F. & STROMINGER J. L. (1976) Isolation and immunologic characterization of a human, B lymphocyte specific, cell surface antigen. J. exp. Med. 144, 98-112. 11. IKEMAN R. L., SULLIVAN A. K., KOSITSKY R., 8/, BARTOK K. (1978) Molecular variants of human Ia-like antigen. Nature 272, 267-268. 12. JANCKILA A. J., LI C. Y., LAM K. W. & YAM L. T. (1978) The cytocbemistry of tartrate-resistant acid phosphatase. Technical considerations. Am. J. Path. 70, 45-55. 13. JONES P. P., MURPHY D. B. & McDEvITT H. O. (1978) Two-gene control of the expression of a murine la antigen. J. exp. Med. 148, 925-939. 14. KATAYAMAI. (1977) Hairy-cell leukemia. New Enol J. Med. 296, 881. 15. KATAYAMAI. & SCHNEIDER G. B. (1977) Further ultrastructural characterization of hairy cells of leukemic reticuloendotheliosis. Am. J. Path. 86, 163-182. 16. KESSLER S. W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. lmmun. 115, 1617-1624. 17. KING J. & LAEMMLIU. (1971) Polypeptides of the tail-fibres of bacteriophage T4. d. Mol. Biol. 62, 465-477. 18. LEMONS. M., PAGANO J. S., UTSINGER P. D. ,g" SINKOVICSJ. G. (1979) Cultured "Hairy cells" infected with Epstein-Barr virus: Evidence for B-lymphocyte origin. Ann. Intern. Med. 90, 54-55. 19. MALENKA D. J., ROGAN K. M., HOWE R. C., NELSON R. S., WROBEL C. J., AHMIED A. R, HUMPHREYS R. E. (1979) Variable expression of membrane proteins among murine lymphoblastoid tumors as seen with sodium dodecylsulfate-polyacrylamide gradient gel electrophoresis of r3SS]methionine-labeled cell membranes. Cancer Res. 39, 4782-4790. 20. MILLER G. 8/: LIPMAN M. (1973) Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natn. Acad. Sci. U.S.A. 70, 190-194. 21. MIYOSHI I.. TSUBOTAT., HIRAKI S., UNO J., NAKAMURA K., HIKITA T., TANAKA T., KIMURA I. & MASUJI H. (1978) Hairy cell leukemia: establishment of a cell line and its characteristics. Nippon Ketsueki Gakkai Zasshi 41,214. 22. NANnA K., SOnAN E. J., BOWLING M. C., BERARD C. W. (1977) Splenic pseudosinuses and hepatic angiomatous lesions. Distinctive features of hairy cell leukemia. Am. J. Clin. Path. 67, 415-426. 23. PERRY L. I., BENACERRAFB. ~" GREENE M. I. (1978) Regulation of the immune response to tumor antigen IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo. J. Immun. 121, 2144-2147.
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24. PINKUSG. S. & SAID J. W. (1977) Profile of intracytoplasmic lysozyme in normal tissue, myeloproliferative disorders, hairy cell leukemia, and other pathologic processes. An immunoperoxidase study of paraffin sections and smears. Am. J. Path. 89, 351-366. 25. POZNEa L. H., D^N~ELSC. A., COOPERJ. A., COHEN H. J., LOC3UEG. L. & CaDgER B. P. (1978) Replication of type I herpes simplex virus in primary cultures of hairy cell leukemic lymphocytes. Am. J. Path. 90, 187-200. 26. RALSTONM. D., D~M~lrn~o J. L., How~ R. C. & HUMrl-mEYS R. E. (1979) Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM Exp. Cell Res. 123, 237-245. 27. ROGAN K. M., FALDETrA T. J., Bo'ro W., AIKEN J. J., DEMAR'rlNO J. L., HOWE R. C., SplRo R. C. & HUMPHREYSR. E. (1978) Heterogeneity in the membrane proteins of human lymphoid cell lines as seen in sodium dodecyl sulfate-polyacrylamide electrophoresis slab gels. Cancer Res. 38, 3604-3610. 28. SALL~N S. E., CHESS L., Fara E. IIl, O'Ba~EN C., NATHAN D. G., STaO~NGER J. L. & SCHLOSSMXNS. F. (1978) Utility of B and T cell specific antisera in the classification of human ieukemias. In Differentiation of Normal and Neoplastic Hematopoietic Cells. (CLARKSONB., MARKSP. A., & TILL J. Eds.), pp. 479-483. Cold Spring Harbor Laboratory Press, New York. 29. S^XON A., STEVENSR. H., QUAN S. G. & GOLDE D. W. (1978) Immunologic characterization of hairy cell leukemias in continuous culture. J. lmmun. 120, 77%782. 30. SCRLOSSMANS. F., CHESSL., HUMPHaEYSR. E. & Sn~O~NGER J. L. (1976) Distribution of h-like molecules on the surface of normal and leukemic human cells. Proc Natn Acad Sci, U.S.A. 741, 1288-1292. 31. SINKOWCSJ. G., WANG C.-H., GY6RgEY F. (1975) Hairy cells in culture. Lancet i, 749-750. 32. SPmo R. C., BOTO W. O., DEMAa'nNO J. L., FALDEI"r^T. J., HOWE R. C., RALSTONM. D., ROGAN K. M., KATAYAM^ I. & HUMPHgEVS R. E. (1980) The diagnosis of subsets of leukemias and lymphomas. In Biomarkers, Genetics and Cancer. (GUmGIS, H. A. & LVNCH H. T., Eds.) Van Nostrand, New York. (In press.) 33. SPlgo R. C., DEMAa~NO J. L., Bo'ro W., LAZARUSH. & HU~PHaEYS R. E. (1979) Comparison of membrane proteins of Burkitt's lymphoma and EBV-transformed B lymphoblast cell lines and of Con A-activated T lymphocytes and T lymphoblast cell lines. Leukemia Res. 3, 315-327. 34. TlC,GES F.-J., v. HEYDENH. W. & WALLERH. D. (1977) Kulturverhalten weisser Blutzellen bei Patienten mit HaarzeiI-Leukiimie in der Diffusionskammer. Blur 35, 103-I 13. 35. UHR J. W., CAPaA J. D., VITgrrA E. S. & COOK R. G. (1979) Organization of the immune response genes. Both subunits of murine I-A and I-E/C molecules are encoded within the I region. Science 206, 292-297. 36. WINC'HEST~gR. J. & ROSS G. (1976) Methods for enumerating lymphocyte populations. In Manual of Clinical Immunology. (ROSE N. R. & FalEDMXN H. Eds.) pp. 64-76. American Society for Microbiology. Washington.
SUBSETS
0145-2126/80/1001~)477102.00/O
OF HAIRY CELL LEUKEMIA UNIQUE
MEMBRANE
DEFINED
BY
PROTEINS
ROBERT C. SPIRO, MOTOHIKO AIBA, ISAO KATAYAMA,* PHILIP P. RAFFA, KIYOSHI SAKAMOTO, DAVID T. PURTILO, JOHN L. SULLIVAN and ROBERT E. HUMPHREYS Departments of Pharmacology, Pathology, Medicine and Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester, MA 01605, U.S.A. (Received 15 May 1980. Accepted 11 June 1980)
Abstract--Leukemic cell membrane proteins of 27 patients with hairy cell leukemia were evaluated by SDS gel eleetrophoresis in order to identify subsets of these patients and to define molecules for isolation and the development of diagnostic reagents. [JSS]methionine metabolic labeling, gel electrophoresis of isolated membranes and fluorography demonstrated about 100 membrane proteins. Variations in the amount of synthesis of two principal proteins, p35 and pl 5 (35,000 and 15,000 dalton proteins respectively),defined, in relative terms, thrc¢ subtypes of hairy cell leukemias: (a) p35*, p15- cells, (b) p35-, p15 ÷ cells, and (c) p35-, p15- cells. At this time, it is not known whether these subsets represent various related disease entities or stages in the progression of one disorder. Although p35 migrated in the gels with one component of the HLA-DR (p23, 30) protein complex, immunoprecipitation studies with anti-p23, 30 serum indicated that it probably was not derived from HLA-DR antigen, which was, in facL synthesized in addition to p35 on all hairy cells which were tested. Cultured lymphoblastoid cell lines established from the peripheral blood of hairy cell leukemia patients were substantially different m membrane protein patterns from the fresh leukemic cells. In particular, p35 and p15 were minimally expressed by cultured cells.
INTRODUCTION VARIATION in survival times among patients with one morphologically defined leukemia has been ascribed to either (a) different degrees of host immune response to control the growth of the malignancy, or (b) the existence of as yet unrecognized leukemia subsets which differ in biological aggressiveness and, thus, in clinical course. The role of the immune system, and particularly of suppressor T lymphocytes, in regulating tumor growth is becoming defined [23]. In addition, cell surface markers have been used to identify biological subsets of leukemias, in some instances with distinct clinical courses and requirements for therapy, e.g. the definition of the p23,30-positive subset of acute lymphoblastic leukemia [28, 30]. With a view towards examining lymphoid malignancies for patterns of membrane proteins which might define subsets, we have refined a technique for examining [JSS]methionine-labeled, leukemic cell membrane proteins by clectrophoresis in SDS polyacrylamide gradient gels [27]. With this method over 100 membrane proteins can be identified. Some proteins are uniquely expressed on T or B cell lines, or at stages of activation of normal lymphocytes ([33], and W. Boto and R. E. Humphreys, unpublished observations). Externally exposed proteins can be defined by su.sceptibility to lactoperoxidase-mediated radioiodination, fucose labeling, or papain cleavage [26]. HLA-A, -B and HLA-DR antigens can be identified by coelectrophoresis *Current address: Department of Pathology. Saitama Medical School. Moro Machi. Saitama, Japan. Abbreviations: EBV. Epstein-Barr virus: HCL. hairy cell leukemia; SDS, sodium dodecyl sulfate. 477
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of immunoprecipitated antigens which are recognized witl~ heteroantisera to p44, 12 and p23, 30, respectively [27, 33]. In applying this method of analysis to lymphoid tumor cell lines, we found a close similarity in membrane protein patterns between a group of Burkitt's lymphoma cell lines and a series of EBV-transformed cells from normal individuals, indicating that in vivo and in vitro transformation with EBV yielded cells frozen at one stage of differentiation [33]. Having established the reproducibility, validity and potential diagnostic value of this method of analysis, we have directed our studies to the leukemic cells of a group of 27 patients with hairy cell leukemia (HCL; leukemic reticuloendotheliosis). The objective of these studies was to describe the membrane protein patterns of freshly obtained leukemic cells in order to identify subsets of patients and to identify unique proteins which might be isolated for development of immunodiagnostic reagents. In addition, cultured cells lines, which were established after EBV infection of the peripheral blood of HCL patients, were examined in order to assess the differences between fresh malignant cells and immortalized cells which were cultured for some time in the absence of hormonal or immunoregulatory influences of the host. MATERIALS AND METHODS Leukemic cells For metabolic labeling with l'3SS]methionine, mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation from the peripheral blood of 27 patients with HCL and one patient (No. l) who presented as a patient with HCL until examination of his spleen, in the course of this study, revealed a well differentiated lymphocytic lymphoma with plasmacytoid features. For all other patients, the diagnosis of HCL was made on the basis of characteristic clinical, hematologic, and pathologic features which included the following: (1) the demonstration of cell surface microvilfi and pseudopods in all 27 cases 1"21,(2) the finding of the ribosome-lamella complex in 14 of 27 patients [15], (3) the presence of positive tartrate-resistant acid phosphatase reactions in 25 of 27 patients [12"l, (4) bone marrow biopsies showing a loose mononuclear cell infiltration with intercellular reticulin fibrosis in 15 of 15 patients from whom such biopsies were obtained [31 and (5) the splenic histopathoiogy demonstrating diffuse mononuclear cell infiltration of the red pulp with formation of pseudosinuses and atropy of the white pulp in 20 of 20 patients [14, 221. In addition, peripheral blood samples from each patient were reevaluated at the time of this study by phase contrast microscopy (demonstrating some to many pathognomonic hairy cells from each patient), electron microscopy and enzyme liistochemistry of tartrate-resistant acid phosphatase and a-naphthyl butyrate esterase activity [91. These examinations were conducted in accordance with the diagnostic principles of multiple investigators [2.3,9. 12, 14, 15, 221. EB V-immortalized cell lines Lymphoblastoid cell lines were established after exposure of Ficoll-Hypaque isolated cells to supernatant suspensions of the B95-8 strain of Epstein-Barr virus (EBV) [20]. Immunofluorescence analysis of these cultured cell lines with reagents specific for IgM, IgD, kappa and lambda determinants were performed [36]. Radiolabeling of cells and SDS electrophoresis of membranes and immunoprecipitated proteins Previously described techniques were followed for labeling cells with [3SS]methionine, isolation and electrophoresis of membranes, preparation of Staphylococcus aureus-bound immunoprecipitates with heteroantisera, and electrophoresis of these precipitates and [14CJacetylated proteins as molecular weight standards [4.16,17,19,26.27]. The preparation and characterization of anti-p44.12 (HLA-A, B antigen and //2-microglobulin) and anti-p23,30 (HLA-DR antigen) sera have been reported [5, I01.
RESULTS Membrane proteins of leukemic cells Variable membrane protein patterns were observed among the ~35S]methioninelabeled, Ficoll-Hypaque-separated, peripheral blood, lymphoid cells of the HCL patients (Fig. l). The membrane protein patterns of the mononucl~r cell preparations reflected principally incorporation into leukemic cells because, in separate experiments, leukemic cells and cultured lymphoblastoid cells were found to incorporate [35SJmethionine at
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FIG. 1, Membrane proteins of hairy cell leukemias, r3~S'imethionine-labclcd, detergent-solubilized membranes of Ficoll-Hypaque-separated lymphoblasts of HCL patients were subjected to electrophoresis in polyacrylamide gradient slab gels. Three subsets of patients could be identified on the basis of relative degrees of synthesis of 35,000 and 15.000 dalton proteins: (a) p35 ÷, p15-, (b) p35-, p15 *, (c) p35-, p15- groups. [J4C']acetylated proteins served as molecular weight standards. HSB and SB are T and B cell lines from one patient (21).
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FIG. 3. Coelectrophoresis of immunopreeipitated p44, 12 (HLA-A, B antigen and /},-microglobulin) and p23, 30 (HLA-DR antigen) with HCL membrane proteins. In order to identify the relationship of HCL membrane proteins to HLA-A, -B and HLA-DR antigens, immunopurified HLA antigens were subjected to electrophoresis. Although p35 ran at the same Rf value as did one component of the HLA-DR complex of 3 proteins (arrows), it was judged not to be derived from HLA-DR antigen for several reasons which are discussed in the text. 481
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F[o. 4. Comparison of freshly obtained leukemic cells with cultured lymphoblasts of the same patients. Membrane proteins of freshly obtained leukemic cells (F) and of cultured lymphoblastold cell lines (C)of individual patients were compared, p35 and p15 were hardly detected on the cultured line and many other differences in expression of minor proteins were found. 482
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rates ranging from 8- to 20-fold the rate of incorporation by normal peripheral blood lymphocytes [33]. The differences in protein band intensities among the various" HCLs fell into two broad categories: (a) variations in levels of synthesis of p35 and p15 (two proteins of 35,000 and 15,000 daltons, respectively) which were prominently synthesized in cells of only some patients, and (b) variations in synthesis of less prominent, or relatively minor, membrane proteins. Four patients (cases 2, 3, 4 and 8) had very high levels of synthesis of p35 (p35 +, p15- type of HCL). Four other patients (cases 1, 9, 13 and 14) had high levels of synthesis of p15 (p35-, p15 + type of HCL). A third group of patients (cases 10, 11 and 12) had low levels of synthesis of p35 and minimal or no apparent synthesis of pl5 (p35-, p15- type of HCL). It should be noted that characterization of these subsets on the basis of p35 synthesis was relative, for although very great amounts of p35 were synthesized by the first subset of HCL, some p35 was in fact observed in membranes of the other two subsets of HCL. Furthermore, a second refinement in nomenclature might be made. That is, "pl 5" constituted 3 proteins about 14,000 15,000 and 16,000 daltons, with only the most prominent one giving its name to this group, In this study, although 28 patients were examined by this methodology, completely satisfactory membrane gels were obtained with calls from only 11 patients. The other 17 patients with low peripheral blood white cell counts (less than 2000 cells/mm 3) did not yield high enough levels of [3SS]methionine incorporation to permit technically adequate gel electrophoresis of their membrane proteins. Consequently, the frequency of patients which were (a) p35 +, p15-, (b) p35-, p15 + and (c) p35-, p15- could not be assessed reliably. There was no correlation between percentages of leukemic cells and white cell counts on the one hand and membrane protein patterns on the other. For example, the white cell counts and percentages of leukemic cells of selected patients were as follows: patient 2, 28.8 x 103WBC, 93% LC (leukemic cells); patient 3, 13.7 x 103WBC, 72Y/o LC; patient 10, 2.3 x 103WBC, 19% LC; patient I, 23.5 x 103 WBC, 41Yo LC; patient 9, 6.9 x 103 WBC, 10% LC. The proteins which were defined in these gels were judged to have been synthesized 90-99% by the leukemic hairy cells because, although normal mononuclear cells were also isolated by the Ficoll-Hypaque gradient, we have shown in previous studies that normal, peripheral blood lymphocytes under these conditions of incorporation, synthesize 1,3SS]methionine into proteins at 5-8% of the rate of leukemic cells i5, 33]. The amounts of the 1,3SS]methionine incorporated into proteins of normal mononuclear cells would be negligible in most instances of this study. Furthermore, in the case of p35-positive patient 2, for example, 93% of the cells were leukemic hairy cells and in pl5-positive patient 1, 41% of the cells were leukemic cells, p35 and p15 thus were clearly synthesized by the leukemic cells of those respective patients. Differences in expression of many minor membrane protein bands were found among the HCL patients but these variations did not form dominant patterns. For example, patient 2 well synthesized a 75,000 dalton protein which was not readily apparent among other patients' cells. Considerable heterogeneity among membrane proteins in the 43,000-48,000 dalton weight range was noted among cells of all patients. Proteins about 28,000 daltons were more extensively synthesized by the four patients' cells which were p35 +, p 15-. Other bands, especially in the 80,000-150,000 dalton range, appeared in cells of a few patients, but their expression did not correlate with the apparent subsets of HCL so clearly defined by levels of synthesis of p35 and p15. As controls for these experiments, the membrane proteins of HSB (from a patient with T cell ALL) and SB (the B lymphoblastoid line from the same patient) I-l], were subjected to electrophoresis in parallel to the HCL samples. Bands which were identified in separate experiments to be HLA-DR antigen (by recognition with anti-p23,30 serum) were present on SB but not on HSB. T cell-related proteins of 18,000, 35,000 and 110,000
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daltons were identified on HSB. [t4CJacetylated proteins were used as molecular weight standards, but the molecular weights of membrane proteins in the Laemmli gel system can only be considered as approximations for reference purposes [271. The function of these many proteins were unknown. However, since p35 migrated at the position of HLA-DR antigen, its relationship to human Ia-like proteins was tested in immunoprecipitation experiments.
Immunoprecipitation of HLA antigens from hairy cell leukemia lymphoblasts lmmunoprecipitates of nonionic detergent-solubilized membrane proteins and heteroantisera to p44,12 (HLA-A, -B antigens and fl2-microglobulin) and to p23,30 (HLA-DR antigen) were obtained with formalinized Staphylococcus aureus. These immunopurified HLA molecules were subjected to electrophoresis in a 147/o polyacrylamide slab gel (Fig. 2). HLA-A, B antigens and fl2-microglobulin were constantly expressed on all HCLs tested by this method. Likewise, HLA-DR antigens were precipitated from each HCL, but a variability in structure among the patients was found. We, and others, have also observed this heterogeneity in apparent molecular weight of HLA-DR components among lymphoblastoid cell lines [:11, 33]. This variability in pattern could reflect structural differences among alleles at HLA-DR loci, as is reported for the I-A and I-E/C loci of the mouse [13, 35]. In order to characterize the relationship of HLA-DR antigen to p35 which was heavily synthesized by HCLs, immunopurified HLA molecules were subjected to electrophoresis along with samples of membrane in a SDS polyacrylamide gradient gel (Fig. 3). HLA-A, -B antigen and flz-microglobulin were identified both in the precipitates and among the membrane proteins. The HLA-DR antigen was resolved into three bands at apparent molecular weights of 32,000, 35,000 and 37,000 daltons, which have been described previously [20]. p35, the extensively synthesized protein on some HCL cells, comigrated with the 35,000 dalton component of the HLA-DR antigen. However, p35 was thought not to be derived from HLA-DR antigen, because a large amount of p35 was not precipitated with anti-p23,30 serum and because the structure of the HLA-DR antigen which was obtained from cells expressing p35 was identical to that from HCL cells not expressing p35 (and to HLA-DR from B lymphoblastoid cell lines, e.g. RAJI, SB, LAZ-007) [5, 27, 33]. If the p35 on some leukemic cells had been part of the HLA-DR complex, then precipitation with anti-p23,30 serum might have recognized more distinctly the unique 35,000 dalton protein which was so heavily synthesized. Further confirmation of the structural difference between p35 of the p35 +, plS= type of HCL and HLA-DR antigen can be obtained through additional chemical studies. Differences in membrane protein patterns between freshly obtained leukemic cells and cultured lymphoblastoid cell linesfrom individual patients The objective of this experiment was to assess whether the membrane protein patterns of freshly-obtained HCL tumor cells were similar to the membrane protein patterns of EBV-transformed, cultured lymphoblastoid cell lines which were established from the peripheral blood of the respective HCL patients. For five patients from whom lymphoblastoid cell lines were established, membrane proteins of cultured lines (C) were subjected to ¢lectrophoresis in wells adjacent to membrane proteins of the respective freshlyobtained HCL tumor cells (F) (Fig. 4). Membrane protein patterns of the EBV-transformed lines from five patients resembled each other and resembled lymphoblastoid cell lines established from normal individuals and from Burkitt lymphomas [33]. The cultured lymphoblastoid cell lines were distinctly different from freshly obtained leukemias. p35 and pl20 disappeared or were greatly reduced in the established cell lines. For patients l and 14, the pl5 protein was absent or was minimally present in the cultured
Membrane proteins of HCL
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cell lines. In addition, differences between cultured cell lines and fresh tumors were seen in many other membrane proteins. For example, p40 and IM6 were more prominently synthesized by cultured lines than by the fresh leukemias. While many of the minor membrane proteins appeared to be the same in the fresh leukemic cells and in the cultured lymphoblastoid cells, the dramatic differences in the expression of several heavily synthesized proteins underscored the finding that fresh ieukemias and the cultured lines were distinctly different. In particular, p35 and plS, which were diagnostic for apparent subsets of hairy cell leukemia, were not well synthesized or were absent from the cultured lymphoblastoid cell lines which were established from patients whose leukemic cells had expressed these respective proteins. Because the five cultured lymphoblastold cell lines were polyclonal for expression of surface IgM and surface IgD and kappa and lambda light chains, they were considered to be derived by EBV-transformation of normal B lymphocytes and not from the malignant leukemic cells.
DISCUSSION The goal of this study was to compare the membrane proteins of tumor cells from a series of patients with hairy cell leukemia (HCL). With a finely resolving methodology of [3~S]methionine labeling and polyacrylamide gel electrophoresis, we have identified membrane protein differences which define three phenotypes of HCL patients. Two heavily synthesized proteins were identified as candidates for isolation and the preparation of immunodiagnostic reagents. In addition, substantial differences in membrane protein patterns were found between fresh leukemic cells and cultured cell lines established from the respective patients. Each of these points can be considered in detail. The membrane protein patterns of different HCL patients varied in their expression of two dominantly synthesized proteins: p35 and plS, proteins of 35,000 and 15,000 daltons, respectively. Three types of cell membrane protein patterns could be identified in relative terms: (a) p35 ÷, p l S - leukemias, (b) p35-, p15 + leukvmias, and (c) p35-, p l S - leukerajas. A close examination of Fig. 1 shows 35,000 dalton protein(s) on all HCL's, although in relative terms, one subtype expressed large amounts (p35 +) and two other types expressed reduced amounts (p35-). The 35,000 dalton proteins on the latter types of cells might not be the same p35 protein which was synthesized by the first subtype of HCL. For example, it could be part of the "underlying" HLA-DR complex. In contrast, p15 + cells showed large amounts of 15,000 dalton protein(s)and p15- cells appeared to synthesize no pl 5. The absolute positivity or negativity of these subsets of leukemias for p35 and p15 can only be established when diagnostic antisera have been prepared to the purified proteins. Such antisera will simplify analyses for these subsets of HCL and will permit screening of other lymphoid malignancies for the presence of p35 and p15. For example, at least one patient with chronic lymphocytic leukemia expressed a 15,000 dalton protein which might cross-react serologically with the p15 found on some HCL patients' cells [32]. Furthermore, patient 1 in this study, who heavily synthesized plS, had a diffuse lymphocytic lymphoma with plasmacytoid features masquerading as hairy cell leukemia. p35 was thought not to be a component of the HLA-DR (Ia antigen-like) complex because (a) large amounts of p35 were not precipitated with anti-p23,30 serum and (b) the HLA-DR proteins which were precipitated from cells which heavily expressed p35, were very similar to the HLA-DR molecules expressed on other cell lines such as B lymphoblastoid cell lines from normal individuals and Burkitt lymphoma cell lines. In addition to observing that p35 is apparently unrelated to HLA-DR antigen, we have, in fact, seen heavy synthesis of several novel 35,000 dalton proteins by some other cells. For example,
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ROBERT C. SPlRO et al.
48-h concanavalin A-activated, E rosette-purified T lymphocytes and 48-h P3HR-I virus superinfected RAJI cells both synthesized relatively large amounts of approximately 35,000 dalton proteins ([33]; W. Boto, D. Casareale, and R. E. Humphreys, unpublished observations). None of these proteins were recognized with anti-p23,30 serum in immunoprecipitation assays. Their relationship to p35 which is seen on HCL cells is unknown at this time, other than their coincidence in size. As with p35, the identity and function of p15 on HCL is unknown, p15 migrated at an R~ value equal to the [:~4C'Jacetylated lysozyme molecular weight standard. However, it probably is not lysozyme because (a) immunoperoxidase studies have failed to find lysozyme on HCL cells in general [:24], (b) patient 1 who had abundant plS on his leukemic cells, had negligible or absent urinary and serum lysozyme, and (c) a rabbit anti-human lysozyme serum (Bio-Rad Labs; catalog No. 170-1572) failed to immunopreo cipitate p15 under conditions at which anti-p44,12 and anti-p23,30 sera recognized their respective antigens. It is not clear at this time whether the phenotypically distinct subsets of HCL, as we have described them here, represent different forms of related diseases or different stages in the progression of one disorder. A cursory review of pathological and clinical findings in these patients does not indicate correlations to the stage, severity, or prognosis of their disease. However, after a year we plan to repeat this study in order to assess possible transitions in phenotype patterns of these patients and to extend our investigation in a systematic fashion to correlations with clinical and pathological findings. The observation that cultured lymphoblastoid lines established from the peripheral blood of patients with HCL failed to express membrane protein patterns of the freshlyobtained malignancies has adverse implications for the preparation of diagnostic reagents. Because the lymphoblastoid lines were polyclonal with respect to expression of surface immunoglobulin, they did not represent the original malignant cell but were derived from EBV-transformed, normal B lymphocytes r36]. Their membrane protein patterns were dramatically different from those of the fresh tumors. In particular, two principal proteins of the tumors, p35 and plS, appeared to be absent from the lymphoblastoid cell lines. Therefore, the cell source for the future isolation of p35 and p15 can only be the fresh leukemic cells. In surveys of fresh leukemias and activated normal T lymphocytes, we have found numerous examples of molecules which are not expressed or are minimally expressed on cultured iymphoblastoid cell lines (I-32, 33]; W. Boto, J. U DeMartino, R. C. Spiro, and R. E. Humphreys, unpublished observations). Consequently, for the isolation of proteins to generate immunodiagnostic reagents, one is forced to turn to fresh leukemic cells or to cultures of activated normal lymphocytes for their respective proteins. One cannot, in fact, take advantage of the large volumes of uniform cells which can be raised with cultured lymphoblastoid lines. Finally, a point of caution is implied in our observation that leukemic cells are different from the EBV-transformed cultured cell line established from the same patient's blood. That is, in the case of other examples of "tumor cell lines," both with respect to HCL [6, 7, 8, 18, 21, 25, 29, 31, 34] and to other tumors, the cultured cells might be quite different from the fresh tumor when membrane protein patterns are analysed by this method. In summary, we have refined a method to examine over 100 membrane proteins of lymphoid malignancies and cell lines, employing r3SS]methionine metabolic labeling and high resolution SDS polyacrylamide gradient gel electrophoresis. Differences were described in membrane proteins among patients with HCL. Three phenotypically different types of patterns were observed on the basis of levels of synthesis of p35 and plS. Although p35 migrated at the weight range of HLA-DR antigen, it was shown to be distinct serologically from that antigen complex. In future studies, we will assess the potential for progression of these subtype patterns of membrane proteins during the
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course of the disease and whether these subtype patterns do, in fact, correlate with clinical and pathological findings and have prognostic implications. Acknowledgements--This study was supported by NCI contract CB 64072 and NCI grant CA 25873 (to R.E.H.), by ACS grant P D T - I M (to I.K.), by NCI grant CA 23561 (to D.T.P.), and by ACS grant IN-129 (to J.L.S.). R.E.H. was the recipient of a Cancer Research Scholar Award from the American Cancer Society, Massachusetts Division, Inc. The expert secretarial assistance of Ms. Lena DeSantis in the preparation of this manuscript is gratefull) acknowledged. We very deeply appreciate the collaboration of many physicians for referring to us their patients, for discussing this project with us and with their patients, and for giving advice and comments.
REFERENCES 1. ADAMSR. A., FLOWERS A. & DAVIS B. J. (1968) Direct implantation and serial transplantation of human acute iymphoblastic leukemia in hamsters. SB-2. Cancer Res. 28, 1121-1125. 2. BOURONCLEB. A. (1979) Leukemic reticuloendotheliosis (hairy cell leukemia). Blood 53, 413-436. 3. BURKE J. A. (1978) The value of the bone-marrow biopsy in the diagnosis of hairy cell leukemia. Am. d. Clin. Path. 70, 876-884. 4. CRAWFORD L. V. ~' GESTELAND R. F. (1978) Synthesis of polyoma proteins in vitro, d. Mol. Biol. 73, 627-634. 5. FALDETTA T. J,, HOWE R. C., ROGAN K. M., SPIRO R. C., KATAYAMAI., PECHET L. & HUMPHREYS R. E. (1978) Demonstration of the internal synthesis of p23,30 by several lymphoid malignancies. Exp. Hemat. 7, 94-104. 6. GOLDE D. W., QUAN S. G. & CLINE M. J. (1976) Hairy cell leukemia. In vitro culture studies. Ann. Intern. Med. 85, 78-79. 7~ GOLD[/ D. W., STEVENS R. H., QUAN S. G. & SAXON A. (1977) Immunoglobulin synthesis in hairy cell leukaemia. Br. J. Haemat. 35, 359-365. 8, HEYDEN H. W., WALLER H. D., PAPE G. R., BEN6HR H. CBR., BRAUN H. J., WILM$ K., RIEBER E. P. & RIETHM/JLLER G. (1976) HaarzelI-Leukiimie I. Klinik, Zytocbemie, Phagnzytosef~ihigkeit yon Haarzellen, Etablierung permanent washsender Zellinien. Dtsch Med. Wochenschr. 101, 9. 9. HIGGY K. E., BURNS G. F. & HAYHOE F. G. J. (1978) Identification of hairy cells of leukemic reticuloendotbeliosis by an esterase method. Br. d. Haemat. 38, 99-106. 10. HUMPHREYS R. E., McCUNE J. M., CHESS L., HERRMANN H. C., MALENKA D. J., MANN D. L., PARHAM P., SCHLOSSMAN S. F. & STROMINGER J. L. (1976) Isolation and immunologic characterization of a human, B lymphocyte specific, cell surface antigen. J. exp. Med. 144, 98-112. 11. IKEMAN R. L., SULLIVAN A. K., KOSITSKY R., 8/, BARTOK K. (1978) Molecular variants of human Ia-like antigen. Nature 272, 267-268. 12. JANCKILA A. J., LI C. Y., LAM K. W. & YAM L. T. (1978) The cytocbemistry of tartrate-resistant acid phosphatase. Technical considerations. Am. J. Path. 70, 45-55. 13. JONES P. P., MURPHY D. B. & McDEvITT H. O. (1978) Two-gene control of the expression of a murine la antigen. J. exp. Med. 148, 925-939. 14. KATAYAMAI. (1977) Hairy-cell leukemia. New Enol J. Med. 296, 881. 15. KATAYAMAI. & SCHNEIDER G. B. (1977) Further ultrastructural characterization of hairy cells of leukemic reticuloendotheliosis. Am. J. Path. 86, 163-182. 16. KESSLER S. W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. lmmun. 115, 1617-1624. 17. KING J. & LAEMMLIU. (1971) Polypeptides of the tail-fibres of bacteriophage T4. d. Mol. Biol. 62, 465-477. 18. LEMONS. M., PAGANO J. S., UTSINGER P. D. ,g" SINKOVICSJ. G. (1979) Cultured "Hairy cells" infected with Epstein-Barr virus: Evidence for B-lymphocyte origin. Ann. Intern. Med. 90, 54-55. 19. MALENKA D. J., ROGAN K. M., HOWE R. C., NELSON R. S., WROBEL C. J., AHMIED A. R, HUMPHREYS R. E. (1979) Variable expression of membrane proteins among murine lymphoblastoid tumors as seen with sodium dodecylsulfate-polyacrylamide gradient gel electrophoresis of r3SS]methionine-labeled cell membranes. Cancer Res. 39, 4782-4790. 20. MILLER G. 8/: LIPMAN M. (1973) Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natn. Acad. Sci. U.S.A. 70, 190-194. 21. MIYOSHI I.. TSUBOTAT., HIRAKI S., UNO J., NAKAMURA K., HIKITA T., TANAKA T., KIMURA I. & MASUJI H. (1978) Hairy cell leukemia: establishment of a cell line and its characteristics. Nippon Ketsueki Gakkai Zasshi 41,214. 22. NANnA K., SOnAN E. J., BOWLING M. C., BERARD C. W. (1977) Splenic pseudosinuses and hepatic angiomatous lesions. Distinctive features of hairy cell leukemia. Am. J. Clin. Path. 67, 415-426. 23. PERRY L. I., BENACERRAFB. ~" GREENE M. I. (1978) Regulation of the immune response to tumor antigen IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo. J. Immun. 121, 2144-2147.
488
ROSERT C. Spmo et al.
24. PINKUSG. S. & SAID J. W. (1977) Profile of intracytoplasmic lysozyme in normal tissue, myeloproliferative disorders, hairy cell leukemia, and other pathologic processes. An immunoperoxidase study of paraffin sections and smears. Am. J. Path. 89, 351-366. 25. POZNEa L. H., D^N~ELSC. A., COOPERJ. A., COHEN H. J., LOC3UEG. L. & CaDgER B. P. (1978) Replication of type I herpes simplex virus in primary cultures of hairy cell leukemic lymphocytes. Am. J. Path. 90, 187-200. 26. RALSTONM. D., D~M~lrn~o J. L., How~ R. C. & HUMrl-mEYS R. E. (1979) Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM Exp. Cell Res. 123, 237-245. 27. ROGAN K. M., FALDETrA T. J., Bo'ro W., AIKEN J. J., DEMAR'rlNO J. L., HOWE R. C., SplRo R. C. & HUMPHREYSR. E. (1978) Heterogeneity in the membrane proteins of human lymphoid cell lines as seen in sodium dodecyl sulfate-polyacrylamide electrophoresis slab gels. Cancer Res. 38, 3604-3610. 28. SALL~N S. E., CHESS L., Fara E. IIl, O'Ba~EN C., NATHAN D. G., STaO~NGER J. L. & SCHLOSSMXNS. F. (1978) Utility of B and T cell specific antisera in the classification of human ieukemias. In Differentiation of Normal and Neoplastic Hematopoietic Cells. (CLARKSONB., MARKSP. A., & TILL J. Eds.), pp. 479-483. Cold Spring Harbor Laboratory Press, New York. 29. S^XON A., STEVENSR. H., QUAN S. G. & GOLDE D. W. (1978) Immunologic characterization of hairy cell leukemias in continuous culture. J. lmmun. 120, 77%782. 30. SCRLOSSMANS. F., CHESSL., HUMPHaEYSR. E. & Sn~O~NGER J. L. (1976) Distribution of h-like molecules on the surface of normal and leukemic human cells. Proc Natn Acad Sci, U.S.A. 741, 1288-1292. 31. SINKOWCSJ. G., WANG C.-H., GY6RgEY F. (1975) Hairy cells in culture. Lancet i, 749-750. 32. SPmo R. C., BOTO W. O., DEMAa'nNO J. L., FALDEI"r^T. J., HOWE R. C., RALSTONM. D., ROGAN K. M., KATAYAM^ I. & HUMPHgEVS R. E. (1980) The diagnosis of subsets of leukemias and lymphomas. In Biomarkers, Genetics and Cancer. (GUmGIS, H. A. & LVNCH H. T., Eds.) Van Nostrand, New York. (In press.) 33. SPlgo R. C., DEMAa~NO J. L., Bo'ro W., LAZARUSH. & HU~PHaEYS R. E. (1979) Comparison of membrane proteins of Burkitt's lymphoma and EBV-transformed B lymphoblast cell lines and of Con A-activated T lymphocytes and T lymphoblast cell lines. Leukemia Res. 3, 315-327. 34. TlC,GES F.-J., v. HEYDENH. W. & WALLERH. D. (1977) Kulturverhalten weisser Blutzellen bei Patienten mit HaarzeiI-Leukiimie in der Diffusionskammer. Blur 35, 103-I 13. 35. UHR J. W., CAPaA J. D., VITgrrA E. S. & COOK R. G. (1979) Organization of the immune response genes. Both subunits of murine I-A and I-E/C molecules are encoded within the I region. Science 206, 292-297. 36. WINC'HEST~gR. J. & ROSS G. (1976) Methods for enumerating lymphocyte populations. In Manual of Clinical Immunology. (ROSE N. R. & FalEDMXN H. Eds.) pp. 64-76. American Society for Microbiology. Washington.