Increased expression of p53 protein in human leukemia cells

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 83, pp , June 1986 Medical Sciences Increased expression of p53 protein in human leukemia cells H. PHILLIP KOEFFLER*, C. MILLER*, M. A. NICOLSONt, J. RANYARD*, AND R. A. BOSSELMANt *Department of Medicine, Division of Hematology/Oncology, University of California Medical Center, Los Angeles, CA 924; and tamgen, 19 Oak Terrace Lane, Thousand Oaks, CA 9132 Communicated by William N. Valentine, February 3, 1986 ABSTRACT We examined synthesis of the cellular phosphoprotein p53 in fresh bone marrow or peripheral blood cells from normal donors and from patients with leukemia, preleukemia, or other hematopoietic disorders. Lysates of cells labeled with [35S]methionine were immunoprecipitated with monoclonal antibodies to p53, and the immunoprecipitates were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Bone marrow or peripheral blood cells from 8 of 33 patients with hematopoietic disorders showed increased p53, seven of the eight occurring in cells of patients with preleukemia or acute myelogenous leukemia. Increased p53 synthesis was not associated with p53 gene amplification, as shown by Southern blot analysis. Synthesis of p53 was not increased in any of nine normal human bone marrow samples or eight normal human peripheral blood granulocyte, macrophage, and lymphocyte samples. The hematopoietic cells of patients in remission or with chronic forms of leukemia did not generally synthesize elevated levels of p53. In addition, we found negligible p53 mrna and protein expression in a variety of human myeloid leukemia lines blocked at different stages of differentiation. Southern blot analysis showed that, except for the HL-6 cells, the p53 gene of the myeloid cell lines was intact. In view of recent evidence implicating p53 in transformation of cultured cells, our results using fresh leukemia cells suggest that p53 may contribute to the phenotype of certain leukemias in vivo. The cellular phosphoprotein p53 was first identified in the nucleus of 3T3 murine cells transformed by simian virus 4 (SV4) (1). Subsequently, this or a similar protein was shown to be present at elevated levels in some but not all cell lines transformed either spontaneously (2) or by a variety of agents, including DNA and RNA tumor viruses (1, 3-7), irradiation (4), and chemical carcinogens (2, 4). Even though the protein is frequently detectable in certain normal cell lines, the level is less than 1% of that occurring in the transformed producer cells. Although the specific function of p53 is not yet clear, recent evidence suggests that it may be involved in the regulation of the cell cycle. Experiments with murine 3T3 cells after serum stimulation showed a 1- to 2-fold increase of p53 expression in late G1 phase (8). In accord with this finding, the DNA synthesis induced in serum-stimulated 3T3 cells could be inhibited by microinjection of anti-p53 monoclonal antibody (9). However, the notion that p53 is associated with rapid cell growth in general may be an oversimplification. Studies of p53 expression in cells derived from mice infected with Friend leukemia virus or polycythemia-inducing Friend virus complex (1) show that although stage one (premalignant) of the induced erythroleukemia is characterized by rapid hyperplasia, these cells do not synthesize high levels of p53 protein. In contrast, stage-two (malignant) cells express high levels of p53. On the other hand, terminal differentiation of murine erythroleukemia and F9 teratocarcinoma cells into nondividing cells The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. results in a marked decrease in concentration of both p53 and its mrna. The association of p53 with malignancy has potentially important clinical implications. We have examined p53 expression in fresh bone marrow or peripheral blood cells from normal human donors and from patients with leukemia, preleukemia, or other hematopoietic disorders, as well as in a diversity of human hematopoietic cell lines. MATERIALS AND METHODS A variety of human hematopoietic cell lines were examined for p53 expression during their exponential growth phase in liquid culture. The myeloid lines included HL-6 (promyelocyte), KG-1 (myeloblasts), U937 (monoblasts), ML-3 (early myelomonoblasts), K562 (early myeloid/erythroid blast cells), A7 [early myeloblasts cloned from the HL-6 line kindly provided by A. Sullivan (McGill University)], and HEL (erythroid/myeloid bipotent cells) (for review ofthe cell lines, see ref. 11). The lymphoid lines included a human T-lymphotropic virus type I (HTLV-I)-infected T-lymphoid cell line known as ME (12) and the Burkitt lymphoma B-lymphoid cell lines Raji and P3HR-1. The SV4-transformed fibroblast line SV-8 was kindly provided by A. Levine (Princeton University). human mononuclear, nonadherent bone marrow blast cells were isolated by density separation (13). Myeloblasts, promyelocytes, and myelocytes represented 7% and erythoid precursors represented 2-25% of the cell population. human peripheral blood granulocytes, monocytes, and B and T lymphocytes were isolated as described (13), and purity of each was greater than 9%. Leukemic, nonadherent mononuclear blast cells were isolated from heparin-treated venous blood or bone marrow by buoyant density centrifugation (13). Cell suspensions contained.86% (median 92%) leukemic blast cells, and the leukemic cells were morphologically and histochemically classified according to criteria developed by the French-American-British Cooperative Group (14). For protein labeling, washed cells were resuspended (5 X 16 per ml) in methionine-free alpha medium containing 1% heat-inactivated, dialyzed fetal bovine serum, and 5 X 16 cells were labeled with [35S]methionine (5,uCi/ml, 14 Ci/mmol; 1 Ci = 37 GBq) for 6 hr. The cells were lysed, and immunoprecipitates were analyzed by NaDodSO4/PAGE and autoradiography as described (15). A small aliquot of each lysate was measured for trichloroacetic acid-precipitable radioactivity. The acute myelogenous and lymphocytic leukemia and the normal and remission bone marrow samples had nearly the same [35S]methionine incorporation per cell (radioactivity in the samples varied by a factor of 2). Likewise, the chronic lymphocytic leukemia and normal peripheral blood samples had nearly the same acid-precipitable [35S]methionine incorporation per cell (again, samples Abbreviations:, acute myelogenous leukemia; HTLV-I, human T-lymphotropic virus type I; SV4, simian virus 4; kb, kilobase(s). 435

2 436 Medical Sciences: Koeffler et al l Proc. Natl. Acad. Sci. USA 83 (19.6) p53 - U~O-69 _~.-46 _ -3 FIG. 1. p53 protein synthesis in human hematopoietic cells. Autoradiogram of a NaDodSO4/polyacrylamide gel shows p53 immunoprecipitated from cells labeled with [35S]methionine for 6 hr: Raji, Burkitt lymphoma B lymphocytes (lane 1); P3HR-1, Burkitt lymphoma B lymphocytes (lane 2); ME, HTLV-I-infected T lymphocytes (lane 3); HEL, erythroleukemia cells (lane 4); nonadherent, low-density human bone marrow cells (lane 5); K562, early erythroblasts (lane 6); HL-6, promyelocytes (lane 7); A7, early myeloblasts (lane 8); U937, monoblasts (lane 9); ML-3, myelomonoblasts (lane 1); KG-1, myeloblasts (lane 11); SV-8, SV4-infected fibroblasts (lane 12). Lane 13: 14C-labeled molecular weight markers (Mr x 1o-3 at right). varied by a factor of 2), but these samples had up to 5% less incorporation than the normal marrow samples. The anti-p53 murine monoclonal antibody used for these studies was PAb 421 (16). Precipitations of several positive and negative samples were also performed with the p53 murine monoclonal antibody PAb 1 (17) with similar results. Antibody titration experiments with PAb 421 and PAb 1 found that 1-1 A1l of antibody per ml of lysate immunoprecipitated equivalent amounts of p53 from 5 x 16 SV-8 cells metabolically labeled (data not shown). All subsequent experiments used 5 Al of the antibody per ml of lysate solution equivalent to 5 x 16 cells. RNA and DNA isolation, electrophoresis, and blot hybridization were carried out as described (18). We used the following DNA clones as hybridization probes: pr4-2 cdna, for p53 analysis (contains the entire coding region for human p53) (19); pnb-1 cdna, for N-myc analysis (2); and the genomic Cla I-EcoRI fragment of the c-myc gene (pmc-41 3 RC) for c-myc analysis (21). RESULTS p53 Expression in Various Human Hematopoietic Cell Lines. We examined p53 synthesis in cells from human leukemic cell lines blocked at different stages of differentiation (Fig. 1). The immunoprecipitate of the SV-8 cells (lane 12) contained the expected doublet of p53 proteins and the complexed large tumor (T) antigen of SV4. Likewise, cells from the Burkitt lymphoma B-lymphocyte lines Raji (lane 1) and P3HR-1 (lane 2) synthesized immunoprecipitable p53. The Raji cells synthesized a p53 doublet, as observed previously (1). We also found prominent p53 synthesis in cells from a human T- lymphocyte line (ME) infected with HTLV-I (lane 3). The HEL cells, a bipotent human erythroid/myeloid hematopoietic cell line (lane 4) synthesized p53. Nonadherent, lowdensity human bone marrow cells (lane 5), mostly (>8%) early myeloid precursors, synthesized a small amount ofp53. As previously observed (6), the molecular weights of ps3 differ slightly among different cell types. No detectable p53 synthesis was observed in the following six human myeloid leukemia cell lines: K562 early erythroblasts (lane 6), HL-6 promyelocytes (lane 7), A7 early myeloblasts (lane 8), U937 monoblasts (lane 9), ML-3 myelomonoblasts (lane 1), and KG-1 myeloblasts (lane 11). Similar results were found in two separate experiments. Total RNA was extracted from 1 of these cell lines and was examined for p53-specific sequences (Fig. 2). The Burkitt lymphoma cells (Raji, lane 1) and the HTLV-Iinfected T lymphocytes (ME, lane 2) contained the 2.8- kilobase (kb) mrna of p53. The Raji cells contained only one mrna that hybridized with the p53 cdna probe, although the p53 protein of Raji was a doublet on the PAGE autoradiogram. The HEL bipotent cells (lane 3) and the normal human marrow precursor cells (lane 4) contained mrna for p53 that could only be seen by a longer exposure of the autoradiogram. All the other myeloid leukemia cell lines had no detectable p53 mrna even after a 1-day exposure of the autoradiogram (lanes 5-1). We performed Southern analysis to determine whether the p53 gene of these myeloid cells was intact. The DNA from HL-6 cells contained only a 2.3-kb p53 hybridizing fragment after digestion with EcoRI (Fig. 3, lane 1). This is consistent with prior findings that HL-6 cells are missing one chromosome 17 and the other has a partial deletion of the 5' side of the p53 gene (). The gene encoding p53 is on human chromosome 17. The other myeloid lines tested (Fig. 3, lanes 2-6) had the expected 14.-kb EcoRI fragments, suggesting that the p53 gene was intact. The KG-1 cells (lane 2) appeared to have less p53 DNA than the cells of the other lines; this has not been pursued further * AP -. e 2.8 Kb- * 4. :.c FIG. 2. p53 RNA accumulation in human hematopoietic cells. Total RNA (2,ug per lane) from Raji (lane 1), ME (lane 2), HEL (lane 3), low-density human bone marrow cells (lane 4), K562 (lane 5), HL-6 (lane 6), A7 (lane 7), U937 (lane 8), ML-3 (lane 9), and KG-1 (lane 1) was electrophoresed, blotted, and probed with p53 cdna pr4-2.

3 Medical Sciences: Koeffler et al. i Kb Kb FIG. 3. p53 DNA in myeloid leukemia cell lines. EcoRI-digested DNA (15 jig per lane) from HL-6 (lane 1), KG-1 (lane 2), HEL (lane 3), K562 (lane 4), U937 (lane 5), and ML-3 (lane 6) was electrophoresed, blotted, and probed with p53 cdna pr4-2. Proc. Natl. Acad. Sci. USA 83 (1986) 437 p53 Protein Synthesis in Cells from Patients with Hematopoietic Malignancies. Mononuclear cells from peripheral blood or marrow were isolated from 33 patients and 17 normal volunteers. Table 1 lists the patients, their disease status at the time of study, and the relative densitometry readings of the p53 synthesis in their cells as compared to p53 synthesis in SV-8 cells, samples of which were electrophoresed in parallel with the samples from the patients. Prior studies showed that SV-8 cells contained large amounts of p53, about 45 pug of p53 per g of total cellular protein (7). human low-density, mononuclear bone marrow cells from 9 donors synthesized -2% (mean.%) of the p53 that is synthesized by the same number of SV-8 cells (Table 1). human macrophages also expressed a small amount of p53, whereas no p53 synthesis was detected in granulocytes or B and T lymphocytes. Preleukemic and acute myelogenous leukemia () cells from the bone marrow or peripheral blood from 7 of 14 patients synthesized 1- to 1-fold more p53 protein than did normal marrow cells. The p53 synthesis in samples of regenerating marrow harvested from 7 of 8 patients placed in remission was equivalent to the synthesis by normal marrow cells. Elevated p53 synthesis occurred in marrow cells from one leukemia patient in remission (no. 13). Synthesis of p53 was low (% of that in SV-8 cells) in relatively mature B and T leukemic lymphoid cells and in marrow from a patient with acquired immune deficiency syndrome (AIDS). The peripheral blood cells from a patient with polycythemia rubra vera (PRV) synthesized elevated levels of p53; this patient developed about 1 year after the p53 determination on Table 1. p53 synthesis in hematopoietic cells from leukemia patients Relative p53 Patient Cell source* Diagnosis/disease statust synthesis, % Patient Cell source* Diagnosis/disease statust 1 Preleukemia CLL 2 Preleukemia CLL 3 Preleukemia 2 27 CLL 4 S (erythroleukemia) (megakaryoblasts), remission, remission, remission, remission, remission, remission CML ALL ALL, remission ALL, remission 46 1; CLL T-cell CLL (Sezary syndrome) 45 A Mac 44 Mac 45 Gran 46 Gran 47 B and T 1 48 B and T 49 L 5 L PRV AIDS and Kaposi sarcoma Marrow hypoplasia Folate deficiency (anemia) Relative p53 synthesis, % Experimental and SV-8 cells (5 x 16 in 1 ml) were cultured with [35S]methionine for 6 hr, and the cell lysate was immunoprecipitated with p53 antibody and subjected to NaDodSO4/PAGE; densitometry readings of the autoradiograms were performed and all data were compared to p53 synthesis of SV-8. Data are expressed as a percent of p53 synthesis by SV-8 cells. *, low-density, nonadherent bone marrow cells;, peripheral blood cells; Mac, macrophages; Gran, granulocytes; B and T, B and T lymphocytes; L, peripheral blood lymphocytes. tcml, chronic myelogenous leukemia; ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; PRV, polycythemia rubra vera; AIDS, acquired immune deficiency syndrome

4 438 Medical Sciences: Koeffler et al q.--m_ -s 4.-I OW 14.5 Kb c-myc 4 ~~~~N-myc FIG. 4. (Upper) p53 DNA in neoplastic cells from patients with myeloid leukemia. EcoRI-digested DNA (1,ug per lane) from human sperm (lane 1) and from myeloid leukemia cells from patient 2 (see Table 1) (lane 2), patient 4 (lane 3), patient 5 (lane 4), patient 9 (lane 5), and three other patients (lanes 6-8) was electrophoresed, blotted, and probed with p53 cdna pr4-2. (Lower) As a control, the filter was stripped of p53 probe and reprobed with c-myc (21) and N-myc (2) DNA. his hematopoietic cells. In total, p53 synthesis was elevated at least 1-fold above normal in marrow cells from 8 of 33 patients. Of the 8 elevated values, 7 occurred in cells of patients with preleukemia or. We examined the DNA from leukemic cells for amplification of the p53 gene (Fig. 4 Upper). Lanes 2-5 represent DNA from leukemic cells from patients 2, 4, 5, and 8, respectively, and as shown in Table 1, the leukemic cells from each of these patients had increased synthesis of p53. Lanes 6-8 represent DNA from the leukemic bone marrow cells of three other patients (p53 synthesis was not examined in these cells). None of the p53-specific hybridizing bands showed gene amplification. Lane 6 does have a weaker hybridizing band and lane 7 has a slightly stronger hybridizing band when compared to germ-line DNA (lane 1); ethidium bromide staining of the gel before transfer of the DNA to the nylon-based paper showed that lane 6 had less and lane 7 had slightly more DNA compared to other lanes (data not shown). This was confirmed by a parallel alteration of the hybridizing bands of lanes 6 and 7 seen on the same blot after removal of the p53 probe and rehybridization with N-myc and c-myc probes (Fig. 4 Lower). Taken together, the data indicate that the increased p53 expression in the patients' cells cannot be explained by amplification of the p53 gene. Proc. Natl. Acad. Sci. USA 83 (1986) DISCUSSION The intracellular concentrations of p53 are elevated in a number of transformed human and rodent cell lines derived from spontaneous or induced tumors (2-6). Transfection studies with rodent cells suggest that p53 expression is related to cellular immortality and may provide a necessary complement for cellular transformation by oncogenes (23-25). We examined levels of p53 synthesis in fresh explants of bone marrow and peripheral blood from 5 human donors and compared them with levels of p53 synthesis in a number of transformed cells, including the SV-8 cell line (Table 1). Seventeen of the donors were clinically normal. The remaining 33 donors exhibited various types of hematopoietic diseases. Levels of p53 synthesis in samples of fresh bone marrow cells from the normal donors were -2% (average.%) oflevels of p53 synthesis observed in SV-8 cells. We considered a greater than 14-fold increase over normal human marrow cells, or 14% of p53 synthesis in SV-8 cells, to be a significant elevation. Of the 33 clinically abnormal samples studied, 8 (nos. 1, 2, 4-7, 9, and 13) showed elevated levels of p53 synthesis, ranging from 14% to 15% of p53 synthesis in SV-8 cells. Seven of the 8 samples showing elevated p53 synthesis (nos. 1, 2, 4-7, and 9) were derived from a group of 12 patients with preleukemia or. Cells from these 7 patients contained p53 at levels 1- to 1-fold those of fresh normal low-density human bone marrow cells. Increased expression of several oncogenes in transformed cells has been associated with amplification of the oncogenes. We did not observe p53 amplification in the DNA from the leukemic patients (Fig. 4). In the present study, considerable diversity existed among preleukemic and patients with respect to p53 synthesis, since 7 samples from this group of 14 patients did not show increased incorporation of [35S]methionine into p53. Clearly, the patients can be categorized into two broad groups, exhibiting either normal or increased synthesis of p53. Since we did not observe elevated expression of p53 in all of the leukemias examined, the continuous elevated expression of p53 is not always essential for the maintenance of transformation. Further studies will be needed to determine whether p53 levels can serve as a marker for distinct subtypes of human leukemia or for other forms of human malignancy. As controls, we examined p53 synthesis in cells from regenerating, remission marrow of leukemia patients. hematopoietic progenitor cells usually are rapidly dividing in the marrow of patients recently put in remission by aggressive therapy. Cells from 7 of 8 regenerating, remission marrows synthesized p53 at levels equivalent to normal human marrow cells. However, cells from one remission marrow (patient 5) synthesized p53 at a 16-fold greater rate than normal marrow. Further observation and studies of this patient, as well as of other patients in remission, may help elucidate the significance of p53 expression with respect to prognosis. The p53 protein data presented in this study are based on a 6-hour metabolic labeling of p53, a period about 6 times the half-life of this protein in HTLV-I-infected human T lymphocytes (1/2-6 min; H.P.K., unpublished observation). Some other transformed cell lines showing elevated levels of p53 exhibited either an extended half-life of the protein or high levels of mrna for p53 (3, 6, 26). We have used relative densitometric values as a measure of incorporation of [35S]methionine into intracellular pools of p53. Values are expressed as a percentage of labeled p53 precipitated from SV-8 cells. However, turnover rates of p53 in the cell lines and tissue samples can vary widely and in most cases are unknown. Moreover, the intracellular pool sizes of both methionine and p53 are unknown. In view of these facts,

5 Medical Sciences: Koeffler et al. densitometric values of p53 synthesis relative to controls can only estimate either rates of synthesis or steady-state levels of this protein. Radioimmunoassay or quantitative immunofluorescence labeling of p53 (7, 26) may provide a more useful method of determining whether p53 levels can serve as a marker of malignancy in distinct subclasses of leukemia, as well as in other forms of neoplasia. We found that cells from most human acute myeloid leukemia lines synthesize negligible p53 mrna and protein (Figs. 1 and 2). These findings are explained in the HL-6 cells, which have a deletion of one human chromosome 17 (chromosome 17 contains the p53 gene) and have a large deletion of the other p53 gene. In contrast, the p53 gene was grossly intact in the other myeloid lines. These myeloid lines are neoplastic by many criteria, including tumor formation in nude mice, and contain the same chromosomal abnormalities present in the leukemic cells from the patient used to establish the cell lines. These data suggest that elevated p53 protein and mrna synthesis are not prerequisites for in vitro immortalization of myeloid leukemic cells. Thus, investigations of only cell lines might not provide a true picture of p53 expression in malignancies in vivo. We wish to express our appreciation to Suzanne Bookstaver and Regina Simon for their secretarial help and to Dr. A. Levine for providing us with monoclonal antibody PAb 421. Supported in part by National Institutes of Health Grants CA2638, CA32737, and CA33936 and Dr. Murray Geisler Leukemia Memorial. H.P.K. is a member of the Jonsson Comprehensive Cancer Center and has a Career Development Award from the National Institutes of Health. 1. Lane, D. P. & Crawford, L. V. (1979) Nature (London) 278, Maltzman, W., Oren, M. & Levine, A. J. (1981) Virology 112, Sarnow, P., Ye Shi, H., Williams, J. & Levine, A. J. (1982) Cell 28, DeLeo, A. B., Jay, G., Appella, E., Dubois, G. C., Law, L. W. & Old, L. J. (1979) Proc. Natl. Acad. Sci. USA 76, Proc. Natl. Acad. Sci. USA 83 (1986) Rotter, V., Boss, M. A. & Baltimore, D. (1981) J. Virol. 38, Crawford, L. (1983) Rev. Exp. Pathol. 25, Thomas, R., Kaplan, L., Reich, N., Lane, D. & Levine, A. (1983) Virology 131, Reich, N. & Levine, A. (1984) Nature (London) 38, Mercer, W. E., Nelson, D., DeLeo, A. B., Old, L. J. & Baserga, R. (1982) Proc. NatI. Acad. Sci. USA 79, Ruscetti, S. & Scolnick, E. (1983) Virology 46, Koeffler, H. P. (1983) Blood 62, Koeffler, H. P., Chen, I. & Golde, D. (1984) Blood 64, Koeffler, H. P., Bar-Eli, M. & Territo, M. (198) J. Clin. Invest. 66, Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A. G., Gralnick, H. R. & Sultan, C. (1976) Br. J. Haematol. 33, Koeffler, H. P., Ranyard, J. & Pertcheck, M. (1985) Blood 65, Harlow, E., Crawford, L. V., Pim, D. C. & Williamson, N. M. (1981) J. Virol. 39, Gurney, E., Harrison, R. & Renno, J. (198) J. Virol. 34, Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 19. Harlow, E., Williamson, N., Ralston, R., Helfman, D. & Adams, T. (1985) Mol. Cell. Biol. 5, Schwab, M., Aletalo, K., Klempnauer, K.-H., Varmus, H. E., Bishop, J. M., Gilbert, F., Brodeur, G., Goldstein, M. & Trent, J. (1983) Nature (London) 35, Favera, R. D., Gelmann, E. P., Martinotti, S., Franchini, G., Papas, T. S., Gallo, R. C. & Wong-Staal, F. (1982) Proc. Natl. Acad. Sci. USA 79, Wolf, D. & Rotter, V. (1985) Proc. Natl. Acad. Sci. USA 82, Eliyalu, D., Raz, A., Gruss, P., Gievol, D. & Oren, M. (1984) Nature (London) 312, Parada, L., Land, H., Weinberg, R., Wolf, D. & Rotter, V. (1984) Nature (London) 312, Jenkins, J., Rodge, K. & Currie, G. (1984) Nature (London) 312, Benchimol, S., Pim, D. & Crawford, L. (1982) EMBO J. 1,