Leukemia Research Vol. 17, No. 6, pp. 523-526, 1993. 0145-2126/93 $6.00 + .00 Printed in Great Britain. © 1993 Pergamon Press Lid

INTERACTION BETWEEN TUMOR NECROSIS FACTOR-re AND HSP 70 IN H U M A N LEUKEMIA CELLS

EVA MOLLER,* REINHOLD MUNKER,t ROLF ISSELSt and WOLFGANG WILMANNS*t *GSF Institute fiir Klinische H~imatologie and tMedizinische Klinik III, Universit~itsklinikum

GrolBhadern der LMU, Miinchen, F.R.G.

(Received 3 January 1993. Revision accepted 12 January 1993)

Abstract--We confirm here by immunoblotting that a cell line resistant to clonal inhibition from tumor necrosis factor (K562) expresses high levels of HSP 70, whereas two sensitive cell lines (HL60 blast and EM3) express low levels. When sensitive cell lines are exposed to tumor necrosis factor, HSP 70 is not generally induced; when tumor necrosis factor is removed, however, HSP 70 is transiently induced. In five bone marrow samples from patients, generally low levels of HSP 70 were detected.

Key words: Tumor necrosis factor, heat shock proteins, human leukemia cells.

I N T R O D U C T I O N

THE FAMILY of heat shock proteins is conserved in evolution and can be induced in mammal ian ceils in response to a variety of environmental stresses, including heat and anoxia [1-3]. Recently, a heat- inducible m e m b e r of the HSP 70 family was detected in human monocytes [4]. Using amplification by poly- merase chain reaction, the uninduced expression of HSP 70 genes by leukemia cells was correlated with resistance to heating [5]. We had earlier investigated the clonal sensitivity of human myeloid leukemia cells to TNF in vitro [6] and now try to establish the correlation between TNF sensitivity and expression of HSP 70 as well as changes of HSP 70 during incubation with TNF.

M A T E R I A L S A N D M E T H O D S

Cell lines The cell lines studied here were obtained from Dr Koef-

tier (UCLA Dept of Medicine, Los Angeles, CA). K 562 is a myeloid leukemia cell with erythroid features derived from a patient in CML blast crisis; EM-3 is a myeloblast cell line and HL60 blast cells are from a late passage of the HI_N) promyelocytic cell line unresponsive to GM-CSF. For comparative purposes, the cell line overexpressing HSP 70 (K 562) was also obtained from two other lab- oratories. All the cell cultures were performed in a humidi- fied incubator at 37°C with 5% CO2. As a cell culture

Abbreviations: TNF, tumor necrosis factor; HSP, heat shock proteins.

Correspondence to: R. Munker, M.D., Medizinische Klinik III. Universit~itsklinikum Groghadern, D 8000 M0nchen 70, F.R.G.

523

medium, RPMI 1640 (Biochrome, Berlin), supplemented with glutamine and penicillin/streptomycin and preselected fetal calf serum, was used. Cells from patients were obtained at diagnostic bone marrow aspirations. Hep- arinized aspirates were separated on Ficoll and washed three times in cell culture medium; all patient samples were handled at 4°C. Recombinant human tumor necrosis factor (TNF) (Genentech Inc.) was kindly provided by Dr Adolph (Boehringer, Vienna). The cells were exposed to the indi- cated concentrations of TNF (1000 or 4000 U/ml) at 37°C for up to 22 h. During this time the cells were maintained in the usual culture conditions. Before exposure to TNF, the cell lines were in active proliferation and >95% viable. After exposure to TNF, the ceils were washed in fresh medium. During and after exposure to TNF, identical aliquots of viable cells were removed and prepared for protein electrophoresis.

SDS-polyacrylamide gel electrophoresis At the specified time points aliquots of cells were taken

for electrophoresis. The cells were washed three times in PBS, suspended in solution buffer (0.01 M Tris-HCl, 5 mM MgC12, pH 7.4) and frozen/thawed four times. For SDS- PAGE the samples were diluted 1 : 1 in 2 × sample buffer (100 mM DTI', 5% SDS, 10% glycerol, 0.06 M Tris-HC1, pH6.8). Equal amounts of protein (100~l/lane) were loaded on the gels. Gel electrophoresis was performed on 10% SDS-polyacrylamide gels in the buffer system of Laemmli [7]. All gels were used for Western blot analysis.

Western blot analysis For immunodetection the proteins, separated as above

by gel electrophoresis, were electrophoretically transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH). The protein blots were blocked with 10% goat serum for 2 h and probed for 2 h with a monoclonal antibody specific for the inducible HSP 70 (-72). This antibody (8F7) belongs to the IgM class and was obtained by immu- nization of rats with extracts of the 72 kd protein from

524 E. MOLLER et al.

TABLE 1. EXPRESSION OF HSP 70 IN 3 MYELOID CELL LINES AND 5 SAMPLES FROM PATIENTS

Cell sample HSP 70 expression TNF-R/S

Cell lines K562 + + + + R HL 60 Blast + S EM-3 (+) S Patients 1,2 (normal) 0 n.d. 3 B.A. (CML, c.p.) 0 S 4K.I. (plasma cell 1.) (+) n.d. 5 K.I. (c-ALL) 0-(+) n.d.

R: resistant in vitro to TNF, S: sensitive in vitro to TNF.

HEK 293 cells [8]. After incubating for 2 h with a second antibody (rabbit-anti-rat IgM) and a peroxidase-con- jugated third antibody (goat-anti-rabbit IgG), the immune complexes were detected, using diamino-benzidine-tetra- hydrochloride (Sigma, Deisenhofen) as a reaction snbstrate. The amount of immunoreactive HSP 70 was quantified by sequentially scanning the blots with a laser densitometer (LKB). The absolute values of the integrated areas under the peaks were plotted vs the time of treatment with resepect to recovery. Statistical significance was deter- mined using Student's t-test.

RESULTS

We show here by immunoblotting that a TNF- resistant leukemia cell line (K562) expresses high levels of HSP 70, whereas two myeloid cell lines sensitive to clonal inhibition by TNF (HL60-blast and EM-3) express low levels of HSP 70. Bone marrow samples from patients with leukemia or other dis- eases also express very low or undetectable HSP 70. These results are given in Table 1. During treatment with TNF, no consistent induction of HSP 70 was observed in sensitive or resistant cell lines or in patient samples. The time studied was 1, 2, 4, and 22 h. In Fig. l(a) and (b) immunoblots are given for the cell lines K562 and EM-3. The only consistent increase of HSP 70 was observed when TNF was washed out and the cell cultures were refed. This increase of immunoreactive HSP 70 was not due to the change of media, since in a control experiment with the EM-3 cell line only a minor increase was noticed (data not shown). The kinetics of these changes are plotted in Fig. 2. As can be seen in Fig. 2, HL60 blasts which basically express more HSP 70 have relatively low induction, whereas the EM-3 cell line which basically expresses almost no HSP 70 has a more pronounced induction of HSP 70.

FIO. l(a): Western blot, lanes 1, 3, 5 and 7 EM-3, lanes 2, 4 and 6 K 562 after 4, 6, 22 and 25 (post-washing out) h; lane 8 molecular-weight marker (70 kd); thick arrow corresponds to HSP 70-specific immunoreactivity, small arrow corresponds to nonspecific cross-reactivity described for AB. (TNF used at 4000 U/ml.) (b): Western blot, lanes 1, 3, 5 and 7 K 562, lanes 2, 4, 6 EM-3 after 0, 1, 2 and 4h (time point only for K 562); lane 8 molecular-weight markers. Thick and small arrows in same position as in

Fig. l(a). (TNF used at 4000 U/ml.)

TNF and HSP 70 in human leukemia cells 525

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FIG. 2. Time course of HSP 70 immunoreactivity during incubation with TNF (1000 U/ml). * Indicates statistically

significant (t < 5%).

DISCUSSION

We confirm here that the myeloid cell line K562 which is relatively resistant to heat shock [5] and which we had found earlier also to be resistant to clonal inhibition by TNF [6] constitutively over- expresses the heat-inducible HSP 70, whereas two other cell lines (EM-3 and HL60 blasts) which we found earlier to be sensitive to TNF [6], express low levels of the inducible HSP 70. Based on these results, we postulate a correlation in myeloid cells between heat-resistance and resistance to TNF, at least as far as cell lines are concerned. The induction of heat-shock proteins is generally regarded as a mechanism which protects mammalian cells against environmental stress [2, 7]. Indeed, two groups found that the induction of stress proteins by heat shock protects mammalian cells from lysis by TNF [9, 10]. Our results show that the treatment of sensitive cells (EM-3, HL60 blasts) with a cytotoxic cytokine does not necessarily lead to the expression of stress pro- teins (inducible HSP 70). We conclude that the treat- ment of cells with TNF-tr and the induction of the heat-shock response are two different phenomena which elicit a differential response. Our experiments are also consistent with data which show that a pre- treatment with TNF or a simultaneous treatment with TNF and increased temperature does not elicit a protective response but leads to increased cyto- toxicity in transformed tumor cell lines [11]. A pat- tern of response similar to our data with TNF was also observed when HL60 cells were treated with interferon [16]. In human myeloid cells not only heat, but also erythrophagocytosis and the treatment with bacterial products can induce HSP 70 [4, 12]. Human T-cells activated by PHA or interleukin-2 also show an increase of HSP 70 after entry into cell-cycle [13]. Our observation that a rebound of HSP 70 occurs

when TNF has been washed out and the cells are refed may in part be cell-cycle-related, since the inducible heat shock proteins are known to be cell- cycle-regulated [3]. Another member of the HSP family, the small heat shock protein HSP 28, was shown to be rapidly phosphorylated but not induced upon incubation with TNF [14]. In different tumor cell lines the expression of manganese superoxide dismutase was described as a protective mechanism against TNF-mediated cytotoxicity [15]. Taken together, mammalian cells have an array of pro- tective mechanisms against environmental stresses. For myeloid leukemia, we demonstrate here that the overexpression of HSP 70 is correlated with TNF- resistance and that the treatment of sensitive cells does not lead to a direct induction of HSP 70. Recently, a high constitutive expression of HSP 90c~ was demonstrated in human acute leukemia [17]; further work will be necessary to study the expression of heat shock proteins (HSP 27, 70 and 90) in further samples from patients with acute and chronic leu- kemias, and to investigate the possible regulation by cytokines like TNF. The involvement of HSP 70 in the protection against TNF mediated cytotoxicity is also underscored by experiments in which the human HSP70 gene was introduced into mouse mye- lomoncytic cells which were made resistant to TNF [181.

Acknowledgements--We thank Ms S. Pander for expert technical assistance, Ms A. Schaaf for editorial help, Dr L. Heine for providing us with the monoclonal antibody 8F7 and the W. Sander Stiftung for financial support.

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