INDUCTION OF DIFFERENTIATION BY IL-6-TYPE CYTOKINES IS IMPAIRED IN MYELOID LEUKAEMIA CELLS UNABLE TO ACTIVATE Stat5a

INDUCTION OF DIFFERENTIATION BY IL-6-TYPE CYTOKINES IS IMPAIRED IN MYELOID LEUKAEMIA CELLS UNABLE TO ACTIVATE Stat5a Roland P. Piekorz and Gertrud M. Hocke The block of differentiation in myeloid leukaemia can be overcome by treatment with a variety of agents including cytokines. Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) induce macrophage differentiation and growth arrest through activation of the Janus kinase (Jak)/signal transducers and activators of transcription (Stat) signal pathway in murine M1 myeloid leukaemia cells. Treatment of various other myeloid leukaemia lines with LIF or IL-6 did not lead to induction of differentiation. Several defects in the cytokine triggered Jak/Stat signal pathway were striking in these lines. They expressed a decreased or undetectable amount of at least one of the components of the specific cytokine receptor complexes. Three lines contained a constitutively activated Jak/Stat signal cascade and in two of them, lines C and BMC-63, this cascade was inducible by treatment with IL-6, despite of a very low density of IL-6-receptors. Apart from the cytokine receptors, additional components of the Jak/Stat signal cascade were altered in these lines. Expression and activation of the transcription factor Stat5a and the tyrosine kinase Jak2 were markedly decreased compared to M1 cells, suggesting a role of activated Stat5a in the induction of differentiation. These results demonstrate a direct correlation between alterations in the Jak/Stat signal pathway and the inability to differentiate after cytokine treatment of myeloid leukaemia cells. 7 1997 Academic Press Limited

Myeloid leukaemia cells are blocked at an early stage of differentiation and proliferate aberrantly.1,2 In culture, leukaemia cells can be induced to differentiate terminally by a variety of agents, including retinoids and cytokines.2,3 These observations led to the concept that induction of differentiation could be used as a therapeutical tool. Differentiation therapy of a human promyelocytic leukaemia (subtype AML-M3) characterized by the chromosomal translocation t(15:17) has been shown to be very efficient. Treatment of the patients with all-trans retinoic acid (ATRA) induced differentiation of malignant cells to mature neutrophils, thereby leading to a remission.4 Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF), members of the family of IL-6-type cytokines5 are capable of inducing differentiation of

From the Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nu¨rnberg, Germany Correspondence to: G. M. Hocke, Chair of Genetics, Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nu¨rnberg, Staudtstrasse 5, D-91058 Erlangen, Germany; E-mail: ghocke.biologie.uni-erlangen.de Received 23 January 1997; accepted for publication 24 March 1997 7 1997 Academic Press Limited 1043–4666/97/090639 + 11 $25.00/0/ck970221 KEY WORDS: myeloid leukaemia/interleukin-6-type cytokines/ induction of differentiation/Stat factors/Jak kinases CYTOKINE, Vol. 9, No. 9 (September), 1997: pp 639–649

M1 cells, a murine myeloid leukaemia cell line, to macrophages.6,7 Simultaneously proliferation of these cells is reduced. Treatment of the murine cell line WEHI-3B and the human line U-937 with IL-6 induces differentiation of the cells to mature macrophages,8 but so far M1 is the only leukaemia line which undergoes terminal differentiation after treatment with LIF. The unresponsiveness of WEHI-3B cells to treatment with LIF is caused by a lack of the ligand binding chain of the LIF receptor (LIF-R). Transfection of the cells with the missing ligand binding chain restored LIF-induced differentiation.9 The main purpose of this work was to investigate whether the responsiveness of M1 cells to LIF and IL-6 was a general property of a majority of myeloid leukaemia cells or unique for M1 cells. The results of this study should clarify the question, whether the signal pathway involved in induction of differentiation by cytokines of the IL-6 family may reveal a therapeutical tool in the treatment of myeloid leukaemia cells. Several independently established cell lines were investigated. One set was generated by transformation of bone marrow cells with Friend Murine Leukaemia Virus.10 The other set was established by transfection of murine bone marrow cells with a retroviral vector containing an expression construct for the chromosomal break point region of the human translocation t(1:19).11 This construct 639

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codes for the chimeric protein E2A/Pbx1, which is correlated with a human pre B-cell leukaemia, but induces myeloid leukaemia in mice.11 Surprisingly, the M1 line was the only cell line in our panel which responded to cytokines of the IL-6 family by an induction of differentiation. This result raised the question of whether the unresponsive lines express minor defects in the signal cascade which could be overcome easily by certain therapeutical tools or whether the defects were so pronounced that the differentiation concept would not be applicable to myeloid leukaemia cells. Therefore, the signal cascade mediating the response to IL-6-type cytokines was investigated. IL-6-type cytokines utilize a common signal pathway, known as the Janus kinase (Jak)/signal transducers and activators of transcription (Stat) pathway.5 The signal starts at the receptor, consisting of a cytokine-specific ligand binding chain and the common signal transducer chain gp130. Binding of the cytokine to its receptor leads to the activation of receptor associated kinases Jak1, Jak2 and Tyk2.12,13 Upon phosphorylation these kinases in turn phosphorylate cytoplasmic transcription factors of the family of Stat proteins at specific tyrosine residues leading to their homo- and heterodimerization.14,15 Dimerized Stat factors translocate to the nucleus and bind to specific response elements of various target genes, thereby modulating their transcription rates. Stat factors 1 to 6 have been identified so far. They are involved in various cytokine mediated responses. Stimulation of different cell types with IL-6-type cytokines leads mainly to the activation of Stat proteins 1 and 3.16–18 Activation of Stats 1 and 3 was observed in M1 cells after treatment with IL-6-type cytokines.16,19 Thereby these Stat factors acquired the ability to bind to specific Stat-binding sites.19,20 Recently, differentiation and growth arrest of M1 cells was shown to be dependent on activation of Stat3.21–23 Although it was demonstrated that Stat3 is essential for the induction of differentiation in M1 cells it was not investigated whether Stat3 was sufficient. We reported that cytokine treated M1 cells contain not only Stats 1 and 3 binding to a confined Stat-binding site, but also Stat5a (R. Piekorz et al., unpublished results). The relative amounts of these factors binding to the Statbinding site changed with increasing time of differentiation. We therefore suggested that the factors involved in DNA-binding additionally to Stat3 might be essential for differentiation of these cells. Recent studies generating Stat1 knock-out mice led to the conclusion that Stat1 is less important for the control of differentiation in the haematopoietic system.24,25 In addition, overexpression of a dominant-negative mutant for Stat1 had no effect on IL-6-mediated

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differentiation of M1 cells.23 Therefore, our attention was focused on the involvement of the third component, the transcription factor Stat5a. Stat5a/MGF (mammary gland factor) was originally isolated as a factor regulating the expression of casein in mammary gland tissue in response to prolactin,26 whereas Stat5b was isolated from rat liver after induction of an acute phase response.27 In mice two highly related Stat5 genes exist coding for Stat5a and Stat5b.28–30 Stat5 proteins mediate the responses induced by various agents like prolactin, cytokines of the granulocyte macrophage-colony stimulating factor (GM-CSF)/IL-3/IL-5 family, erythropoietin (EPO) or growth hormone.26,28,29,31,32 Conflicting data exist about the function of Stat5 in the haematopoietic system. In UT7 and FDCP-1 leukaemia cells the activation of Stat5 by thrombopoietin and EPO, respectively, was correlated with an increased rate of proliferation.33,34 On the other hand, activation of Stat5a by treatment of U-937 cells with GM-CSF, interferons or phorbol esters was correlated with terminal differentiation of these myeloid leukaemia cells.35,36 This indicated a role of Stat5a in differentiation processes of leukaemia cells. Additional induction of differentiation of M1 cells by IL-6-type cytokines was due to the involvement of the Jak/Stat pathway21–23 and was paralleled by activation of the transcription factors Stats 1, 3 and 5a (R. Piekorz et al., unpublished results). Based on the data obtained from M1 cells, we asked whether the unresponsiveness of the other cell lines correlated with a loss or an inadequate expression and activation of one or more of the components of the Jak/Stat signal pathway.

RESULTS M1 cells, but none of the other myeloid leukaemia cells respond by induction of differentiation and reduction of proliferation to treatment with LIF or IL-6 Proliferation rates of eight myeloid leukaemia cell lines were measured for both untreated cells and cells treated with various inducers of haematopoietic differentiation. Only M1 cells displayed a significant reduction of cell numbers after a four-day treatment with LIF or IL-6 (Table 1). None of the other lines responded to these cytokines, but their proliferation was clearly inhibited by various other inducers of differentiation (Table 1). The lines under study reacted with characteristic differences to treatment with various differentiation promoting agents. Lines A/HF1 and L/HF8 responded only to ATRA and dexamethasone (Dex), whereas A201.20 cells were responsive to all of the agents tested, except

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TABLE 1. Only M1 cells respond by a reduction of proliferation to treatment with LIF or IL-6 Differentiation agent

M1

A/HF1

L/HF8

C

A201.20

G/HF4

BMC-9

BMC-63

LIF IL-6 ATRA TPA Vit.D3 LPS Dex

35 17 74 128 70 32 97

107 107 9 123 96 123 32

107 99 17 95 100 89 78

115 100 119 85 108 61 107

108 111 7 56 74 43 43

112 141 49 90 85 112 105

97 100 13 109 102 92 91

95 90 37 103 116 102 75

Proliferation of various myeloid cell lines in the presence of known inducers of differentiation was determined. Lines were treated for four days with one of the following agents as described in Materials and Methods: LIF, IL-6, ATRA, TPA, Vit.D3 , LPS and Dex. The number of viable cells was then determined by trypan blue exclusion and expressed as a fraction of the number of untreated cells. Values given in the table were calculated as percentage of untreated control cells. Data were measured in duplicate and are representative of two experiments.

for cytokines of the IL-6 family. All lines responded at least to one of the differentiation-promoting agents, not only with reduced proliferation, but also with induction of differentiation. Differentiation was measured by the increase of phagocytic activity and was calculated exemplarily for two cell lines. After treatment of BMC-9 and C cells with ATRA or lipopolysaccharide (LPS), respectively, the amount of phagocytic cells increased to a similar degree as for LIF-treated M1 cells (Fig. 1). Thus, although all lines of the panel had the potential to differentiate, no induction of differentiation was observed after their incubation with LIF or IL-6, except for M1 cells. The question was asked whether this unresponsiveness was due to a deficiency in a major component of the Jak/Stat signal cascade. This pathway was demonstrated to be critical for IL-6-/LIF-triggered induction of differentiation of M1 cells.21–23 The first components analysed were the high affinity receptors for IL-6 and LIF.

Unresponsive myeloid leukaemia lines express reduced levels of mRNA for at least one component of the IL-6 - or LIF-receptor complex High-affinity receptors for IL-6 and LIF consist of specific ligand binding chains (LIF-R, IL-6R) and the common signal transducer gp130. All lines except M1 lacked the mRNA for the LIF-R as revealed by reverse transcriptase polymerase chain reaction (RT-PCR; Fig. 2) and Northern blot studies (data not shown). The mRNA for the IL-6R was expressed in all lines except A/HF1, but in four of six lines the expression levels were very low (Fig. 3A and Table 2). The L/HF8 and C lines expressed an IL-6R mRNA with a different electrophoretic mobility, probably due to structural alteration (Fig. 3A). In two lines, A201.20 and BMC-9, mRNAs for gp130 were not detectable and in all other lines the expression levels were reduced (Fig. 3B and Table 2). The lack of one essential receptor component provides a satisfactory explanation for the unresponsiveness of all lines to treatment with LIF and of lines A/HF1, A201.20 and BMC-9 to treatment with IL-6. The other four lines

80 Phagocytic cells (%)

ATRA 60

LPS

0

1

2

3

4

5

6

7

8

9

LIF

40

20

0 Figure 1.

Figure 2. Only M1 cells contain the ligand binding chain of the LIF-receptor.

M1

BMC-9

C

Myeloid leukaemia cells are capable to differentiate.

Terminal differentiation was analysed by measuring phagocytic activity. Myeloid leukaemia cells were either kept untreated (q) or incubated with the indicated agents for 4 days (Q). Cell samples were then analysed and the percentage of cells ingesting latex beads was determined. The result of one representative experiment is shown.

The occurence of the mRNA for LIF-R was analysed by RT-PCR. Primers specific for the extracellular portion of murine LIF-R were used (Materials and Methods). The length of the amplified fragment was 512 bp (upper panel) and the specificity of the amplified product was verified by sequence analysis. PCR with primers for b-actin was performed in parallel as control (202 bp; lower panel). Tracks: (0) no template, (1) mouse liver, (2–9) myeloid cell lines M1, A/HF1, L/HF8, G/HF4, BMC-63, A201.20, BMC-9 and C, respectively.

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G/HF4

L/HF8

had reduced mRNA levels for both components of the IL-6 receptor. The question was asked whether the reduced levels could be the reason for the unresponsiveness towards IL-6.

M1

A

M1

The mRNA levels of IL-6R and the signal transducer gp130 were analysed in several Northern blot experiments similar to the one shown in Fig. 3. Relative amounts of mRNAs were determined by 2D-densitometry and expressed as fraction of the expression in M1 cells (100%). nd, mRNA not detectable by Northern blot analysis.

C

100 87 nd 9 18 73 16 7

BMC-9

100 27 7 54 nd nd 3 15

A201.20

M1 L/HF8 A/HF1 BMC-63 A201.20 BMC-9 C G/HF4

BMC-63

IL-6R

A/HF1

gp130

L/HF8

Cell line

increase in the intensity of complex II (Fig. 4A, tracks 5 and 6). Although C cells contained over 30-fold less gp130 mRNA and more than 6-fold less IL-6R mRNA than M1 cells, these levels were sufficient to confer cytokine response. BMC-63 cells expressing only half of the amount of mRNA for gp130 and 10-fold less of the IL-6R than M1 cells, responded to treatment with IL-6 by an increase in DNA-binding capacity similar to the result gained for C cells (R. Piekorz and G. Hocke, unpublished observation). Nevertheless, although C and BMC-63 cells contained a cytokineinducible complex II no functional effects as inhibition of proliferation or increase in the number of phagocytic cells were detectable.

M1

TABLE 2. Myeloid leukaemia cells contain varying amounts of the mRNAs for the IL-6 receptor

Stat factors are activated in C cells after treatment with IL-6 but not with LIF Nuclear extracts from C cells incubated with LIF showed no increase in the intensity of complex II compared to uninduced C cells (Fig. 4A, tracks 3 and 4). The same result was gained after treatment of BMC-9 cells with IL-6 (data not shown). These results were consistent with the lack of the LIF-R or gp130 in C and BMC-9 cells, respectively (Fig. 2 and Table 2). Nevertheless, treatment of C cells with IL-6 led to an

L/HF8

BMC-63

B

C

Activation of Stat factors has been demonstrated routinely by electrophoretic mobility shift assays (EMSA) using the oligonucleotide TB2 as probe, which consists of a dimer of a Stat-binding site.37–39 Using the TB2 probe a specific protein–DNA complex, complex II, was formed with nuclear proteins from LIF-treated M1 cells, but not with corresponding extracts from untreated cells (Fig. 4A, tracks 1 and 2). A faint complex II was already detectable with extracts from untreated C, BMC-63 and BMC-9 cells (Fig. 4A, tracks 5, 7 and 8, respectively) but none of the other cell lines (R. Piekorz and G. Hocke, unpublished data). The DNA-binding specificities of nuclear proteins from C, BMC-9 and BMC-63 cells were similar to M1 cells. This was exemplarily demonstrated for nuclear extracts of C cells by EMSA-competition studies with specific and mutated oligonucleotides representing the Stat-binding site (Fig. 4B).

M1

Cell lines C, BMC-63 and BMC-9 contain constitutively activated Stat factors

Figure 3. The amounts of mRNAs of the components of the IL-6 receptor are strongly decreased in murine myeloid leukaemia lines. Accumulation of the mRNAs for IL-6R (A) and the signal transducer gp130 (B) from various myeloid leukaemia lines was analysed by Northern blot experiments. Poly A+-enriched RNA (3 mg) was loaded per track. Filters were subsequently hybridized with probes for murine IL-6R (A) or murine gp130 (B) (upper panels) and GAPDH as loading control (lower panels). The length of the mRNA for IL-6R was 5.3 kb in M1, BMC-9 and A201.20 cells and 5.7 kb in C and L/HF8 cells. The lengths of the gp130 messages were 8.4 kb and 6.4 kb, respectively.

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A

1

2

3

4

5

6

7

8

II

the amounts were reduced in C and BMC-63 cells. Additionally, only marginal levels of the tyrosine kinase Jak2 were detectable in these cells, whereas BMC-9 cells contained comparable amounts to

A

M1

C

4.1 kb B

1

2

3

4

5

3.4 kb

Stat1

6

2.9 kb II

4.3 kb

Stat3

II 4.6 kb Stat5 3.7 kb Figure 4. Nuclear extracts from cytokine treated M1 and C cells form complex II with the Stat-binding site.

GAPDH

B

Similarly to M1 cells, three different variants of Stat1 mRNA (4.1, 3.4 and 2.9 kb), two variants of Stat5 mRNA (4.6 and 3.7 kb) and one species of Stat3 mRNA (4.3 kb) were detected in C cells (Fig. 5A). The concentrations of the mRNAs for Stat factors 1 and 5, but not 3, were significantly lower in the unresponsive cells than in M1 cells (Fig. 5B). All three Stat proteins were present in C, BMC-63 and BMC-9 cells (Fig. 6). BMC-9 cells contained similar levels of Stat proteins 1, 3 and 5a as M1 cells, whereas

Stat3

75

Stat5 Stat1

50

3.7 kb

4.6 kb

–

4.3 kb

–

2.9 kb

3.4 kb

25 0

Cytokine-unresponsive cell lines contain low amounts of Stat factors in the nuclei

1.4 kb

100

4.1 kb

Relative amounts of Stat mRNAs in C cells (percent of M1 cells)

A: EMSA experiments with nuclear extracts of myeloid leukaemia cells and the radiolabelled probe TB2 (Materials and Methods) were performed. Nuclear extracts of various myeloid leukaemia cells untreated or treated with IL-6 or LIF were prepared and the DNA-binding capacity in EMSA experiments compared. Tracks: (1) M1, untreated; (2) M1, LIF-treatment for 15 min; (3–6) C cells: (3, 5) no treatment, (4) treatment with LIF for 1 h, (6) treatment with IL-6 for 15 min; (7) BMC-9 cells, untreated; (8) BMC-63 cells, untreated. B: Sequence specificity of proteins binding to TB2, a Stat-binding site. EMSA experiments were performed with nuclear extracts from cytokine treated M1 (upper panel) and C cells (lower panel) and 100-fold molar excess of double stranded oligonucleotides:37 (1) no competitor; (2) TB1, representing one copy of the core element of the Stat-binding site; (3) mTB1, mutant TB1 with the central part of the core-element destroyed by mutagenesis; (4) TB3, representing one copy of the core homology element of the Stat-binding site; (5) mTB3, mutant TB3 with the central part of the core homology element destroyed by mutagenesis; (6) CA1, one copy of the core homology and core elements in their natural configuration. II: complex II.

Figure 5. mRNA concentrations for Stats 1 and 5 are decreased in C cells compared to M1 cells. A: Northern blot analysis was performed using 3 mg of polyA+enriched RNA from M1 or C cells and specific cDNA fragments for murine Stats 1, 3 and 5 as radiolabelled probes. Hybridization with a cDNA probe for GAPDH was used as loading control. Sizes of specific signals are indicated on the right. B: Relative concentrations of Stat factor mRNAs of C cells compared to M1 cells. Intensities were evaluated by 2D-densitometry of autoradiographs (A) and corrected for constant loading by normalizing the values to the GAPDH signals. Numerical values given are the ratios of the signals from C cells divided by the corresponding signals from M1 cells.

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1

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2

3

4

was significantly lower in BMC-63 and C cells. No Stat5a was detectable in nuclei of these cells after treatment with IL-6 (Fig. 7, tracks 5 and 7) and the amounts of Stat3 and Stat1 were reduced compared to the cytoplasmic fraction. Only BMC-9 cells contained high levels of Stat factors both in the cytoplasm and in the nuclei (Fig. 7, tracks 3 and 4).

S1α/β

S3

Cytokine-treated M1 and C cells contain different spectra of activated Stat factors

S5a

Jak2

Figure 6. C and BMC-63 cells contain low concentrations of Stat factors and the tyrosine kinase Jak2. Whole cell extracts were prepared from the myeloid lines under study and analysed by Western blotting. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Specific antibodies against Stat1a/1b (S1a, S1b), Stat3 (S3), Stat5a (S5a) and Jak2 were used as indicated. Cell lines: (1) M1, (2) C, (3) BMC-63 and (4) BMC-9.

M1 cells (Fig. 6). To judge the significance of this observation it was important to study not only the amount of Stat factors but also their state of activation. Stat factors can enter the nuclei only after tyrosine phosphorylation.15 Therefore, the distribution of Stat factors in the nuclei and cytoplasm was studied. As expected, the amount of Stat proteins was higher in the nuclei of LIF-treated M1 cells than in the cytoplasmatic fraction (Fig. 7). In the other cell lines the quantitative compartimentation of Stat factors was completely different. They displayed Stat proteins predominantly in the cytoplasm, even after treatment of the cells with cytokines. This result predicted a defect in the mechanism of tyrosine phosphorylation. Therefore it was not surprising that the amount of Jak2 1

2

3

4

5

6

7

8 S1α/β

S3

S5a

Figure 7. nuclei.

IL-6-resistant cells contain low levels of Stat factors in the

Western blot experiments were performed with nuclear (odd tracks) and cytoplasmic extracts (even tracks) of M1 cells treated with LIF (tracks 1 and 2) and the following lines treated with IL-6: BMC-9 (tracks 3 and 4); C (tracks 5 and 6); and BMC-63 (tracks 7 and 8). Specific antibodies against Stat1a/1b (S1a, S1b), Stat3 (S3) and Stat5a (S5a) were used as indicated.

The composition of Stat factors involved in complex II formation of LIF-treated M1 cells and IL-6-treated C cells was compared by EMSA-supershift experiments. Addition of anti-Stat3 antibodies to the binding reactions caused the disappearance of complex II and the occurence of complexes migrating with slower mobility, so called ‘‘supershifted’’ complexes, both with nuclear extracts of LIF-treated M1 cells (Fig. 8A, track 4) and IL-6-treated C cells (Fig. 8B, track 4). Anti-Stat1a and anti-Stat5a antibodies had no effect on the mobility of complex II from C cells (Fig. 8B, tracks 3 and 5, respectively), while these antibodies were reactive with nuclear extracts from LIF-treated M1 cells (Fig. 8A, tracks 3 and 7). There was no difference in the spectra of activated Stat proteins of LIF- or IL-6-treated M1 cells (R. Piekorz et al., unpublished results). In contrast to cytokine-treated M1 cells, complex II of IL-6-treated C cells contained no Stat1a and Stat5a. In nuclear extracts from BMC-9 and BMC-63 cells, only Stat3 could be proved to participate in formation of complex II (R. Piekorz and G. Hocke, unpublished data).

DISCUSSION The main findings of this study were: (1) None of the panel of murine myeloid leukaemia cell lines tested, except M1 cells, responded by an induction of differentiation to treatment with IL-6 or LIF. (2) All of the unresponsive lines lacked the specific LIF-receptor ligand binding chain. The mRNA levels of the specific IL-6-receptor ligand binding chain and the signal transducer chain gp130 were reduced compared to M1 cells. (3) Three lines, C, BMC-9 and BMC-63, contained constitutively activated Stat factors unable to induce differentiation in these cells. (4) Although the levels of the mRNAs for the IL-6-receptor components were low in C and BMC-63 cells the Jak/Stat signal cascade was activated by treatment of the cells with IL-6. No functional effects—neither differentiation nor growth arrest— were achieved.

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A

1

2

3

4

5

6

7 S

II

B

1

2

3

4

5

S

S II

Figure 8. A different set of Stat factors is activated in LIF-treated M1 cells compared to IL-6-treated C cells. A: Complex II of LIF-treated M1 cells contains Stat1a, Stat3 and Stat5a. Nuclear extracts from M1 cells treated with LIF (tracks 1 and 5) were preincubated with control antibodies (tracks 2 and 6) or antibodies against Stat1a (track 3), Stat3 (track 4) or Stat5a/MGF (track 7). Extracts were then analysed in EMSA experiments with the radiolabelled probe TB2 as in Figure 4. The final antibody dilutions were 1:20. Tracks 5 to 7, longer exposure of the autoradiogram compared to tracks 1–4. B: Stat1a and Stat5a are not included in complex II formed with nuclear extracts from IL-6-treated C cells. Nuclear protein extracts were isolated from C cells treated with murine IL-6. Proteins were preincubated with antibodies specific for Stat factors and analysed as in A. Tracks: (1) no antibodies, (2) control antibodies, (3–5) antibodies against Stat1a (3), Stat3 (4) and Stat5a/MGF (5). II, complex II; S, shifted complexes.

(5) The inducible and constitutive protein–DNA binding in lines C, BMC-9 and BMC-63 was due to activated Stat3. Stat1 and Stat5a were not activated to bind DNA. This was in clear contrast to cytokinetreated M1 cells, in which activation of Stat3 was accompanied by DNA-binding of Stats 1 and 5a. The differentiation behaviour of a panel of myeloid leukaemia cell lines of independent origin was investigated regarding their response to IL-6-type cytokines. None of the cell lines, except the well-known line M1, responded to treatment with IL-6 or LIF by an induction of differentiation. However, incubation of the cells with other differentiation promoting agents as ATRA or LPS led to differentiation and growth arrest (Table 1 and Fig. 1). In contrast, M1 cells did not undergo terminal differentiation after exposure to these agents although their proliferation was inhibited

(Table 1). The induction of differentiation by IL-6 and LIF in M1 cells was strictly dependent on the activation of the Jak/Stat pathway.21–23 Here we demonstrated that the missing induction of differentiation in the other cell lines was linked to various defects in the Jak/Stat pathway. The response of various cells to certain cytokines is often regulated at the level of expression of specific receptors. Thus, the finding of a reduced or even missing expression of at least one crucial part of the cytokine receptor complexes in the cells under study was not unexpected. The missing expression of the LIF-R (Fig. 2) explains the unresponsiveness to treatment with LIF. Nevertheless, even low levels of mRNA for gp130 and of the IL-6R were sufficient to confer IL-6-induced activation of the Jak/Stat signal cascade in C and BMC-63 cells (Fig. 4A). In contrast to M1 cells, the activation of the Jak/Stat signal cascade did not lead to induction of differentiation in these cells. Possible explanations include defect(s) in the target genes of the Jak/Stat cascade and/or in the cascade itself. We focused our search on the second hypothesis and started looking at the endpoint of the cascade, the Stat factors. Interestingly, the DNA-binding activity, revealed as complex II, was constitutively present in C, BMC-63 and BMC-9 cells (Fig. 4A) and contained Stat3 (Fig. 8B). The reason for the constitutive activation of Stat3 has not been further investigated. The presence of an activated protein tyrosine kinase, for example Src, which can associate with Stat3 and activate it,40 would offer a possible explanation for this finding. Constitutive activation of Stat proteins in primary lymphoid and myeloid leukaemia cells was correlated to the malignant proliferation of the cells.41,42 Stat proteins are normally activated by tyrosine phosphorylation through Jak kinases as demonstrated for Jak2 and Stat5a.43 Jak2 was constitutively activated in acute lymphoblastic leukaemia (ALL) cells and inhibition of Jak2 reduced the proliferation rate of these leukaemia cells.44 The activation of Jak2 correlated in this case with uncontrolled proliferation. These observations are strong hints for a link between dysregulation of either tyrosine kinases or activation of transcription factors of the Jak/Stat pathway and the oncogenic potential of leukaemic cells. In BMC-9 cells high levels of the tyrosine kinase Jak2 as well as of all Stat factors were striking. Therefore, BMC-9 might be another member of cell lines in which dysregulated tyrosine phosphorylation of Stat factors correlates with the oncogenic potential. In contrast to these reports in which the Jak/Stat signal cascade was correlated with proliferation, in M1 cells activation of the Jak/Stat signal cascade by treatment of the cells with IL-6-type cytokines correlated with induction of differentiation (R. Piekorz et al., unpublished results).21–23 Lines C and

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BMC-63 also responded to treatment with IL-6 by an increased activation of Stat factors. However, no induction of differentiation or reduction of proliferation was achieved. Comparison of the signal pathway of these cell lines with M1 cells should finally reveal the critical component which directs the cells either to differentiation or proliferation. Stat factors 1, 3 and 5a were detected in the unresponsive lines, but in far lower nuclear concentrations than in M1 cells (Fig. 7). Although incubation with IL-6 led to an increase in the intensity of complex II in C and BMC-63 cells, the composition of Stat factors differed compared to M1 cells. In contrast to M1 cells, in which Stats 1, 3 and 5a were present (Fig. 8A), complex II of unresponsive cells contained only Stat3 (Fig. 8B). In M1 cells, the relative amounts of Stat factors responsible for the formation of complex II, especially Stat5a, changed during the course of induced differentiation (R. Piekorz et al., unpublished results). Although it was demonstrated that Stat3 is necessary to induce differentiation,21–23 our data imply that Stat3 may not be sufficient. We propose that additional factors are needed to induce terminal differentiation of myeloid leukaemia cells. Several studies23–25 confirmed our suggestion that Stat1 might be less important for the control of differentiation in the haematopoietic system. Therefore our attention was drawn to the third known protein component of complex II: Stat5a. All cells unable to differentiate had one feature in common: They contained no activated Stat5a capable of binding to the Stat-binding site. A critical function of Stat5a in the interferonand phorbol ester-induced differentiation of human monoblastic U-937 leukaemia cells was recently demonstrated.36 Stat factors need to be phosphorylated to bind DNA.14 Stat5a was specifically phosphorylated by the tyrosine kinase Jak2,43 focusing our attention to this particular kinase. Jak2 was contained in high amounts in M1 and BMC-9 cells, but only marginal levels were detected in cellular extracts of C and BMC-63 cells (Fig. 6). If Jak2 specifically phosphorylates Stat5a and Stat5a is essential for transcriptional activation, then the decreased expression of Jak2 could explain the functional unresponsiveness of C and BMC-63 cells to treatment with IL-6. A similar connection was recently demonstrated in Drosophila sp. Both gain of function mutations of a homologue to Jak2 and loss of function mutations of a homologue to Stat5 were correlated with a phenotype resembling a leukaemialike haematopoietic defect in Drosophila sp.45–47 Interestingly, although high concentrations of Stat5a and Jak2 were found in BMC-9 cells (Fig. 6 and 7), no binding of Stat5a to the Stat-binding site could be demonstrated. This might be due to a different activation potential in this cell line or represent an

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additional defect in the Jak/Stat signal cascade. To achieve maximal DNA-binding capacity and transcriptional activation by Stat factors 1 and 3 serine phophorylation of these proteins seems to be required.48,49 Serine/threonine phosphorylated species of Stats 1, 3 and 5 have been observed in various cell types, indicating that this modification is a general property of Stat proteins.19,48–51 Thus, the lack of functional responses in the lines of our panel could possibly be due to defects in serine phosphorylation of Stat factors. Especially, the properties of the cell line BMC-9, which contains high amounts of Stat5a and Jak2, but can not respond to cytokine treatment due to a lack of the signal transducer chain gp130, imply that additional modifications induced by cytokine treatment are responsible for the functional responsiveness of cells. These aspects remain to be addressed experimentally. The comparison of a series of myeloid cell lines led to the conclusion that all lines of the panel investigated had several defects in the Jak/Stat signal cascade. Nevertheless, all unresponsive lines had one defect in common: compared to the only responsive cell line M1, they contained no activated Stat5a. If the lack of activated Stat5a in the unresponsive cell lines is not only correlated with, but actually the cause of the missing differentiation, then a therapeutic concept may become useful: modulation of the activity of kinases involved in phosphorylation of Stat5a could potentially alter the differentiation status of leukaemic cells. Therefore, induction of differentiation of myeloid leukaemia cells by increasing the amount of activated Stat factors may still offer a viable therapeutic option for the future.

MATERIALS AND METHODS Cell culture and reagents The murine myelomonocytic cell line M1 (American Type Culture Collection, Rockville, MD) and murine myeloblastic/promyelocytic cell lines A/HF1, G/HF4 and L/HF811 were grown in RPMI medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum (BioWhittaker, Verviers, Belgium), 2 mM -glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and, except for M1 cells, 30 mM 2-mercaptoethanol. The murine myeloblastic cell lines A201.20, C, BMC-9 and BMC-6310 were maintained in aMEM medium (BioWhittaker, Verviers, Belgium) supplemented with 6% fetal calf serum, 2 mM -glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. The supernatant of the stably transfected CHO-LIF cell line (Genetics Institute, Cambridge) was used as a source of human LIF at a concentration of 3 ng/ml. Cells were treated either with 50 units/ml human IL-6 (T10 units, Genetics Institute, Cambridge, MA) or 10 ng/ml murine IL-6 (Gibco, Eggenstein, Germany). Cells were incubated for 96 h with the following differentiation promoting agents: 1 mM

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ATRA, 100 ng/ml LPS (Type 026:B6 from Escherichia coli), 100 nM Dex, 1 nM cholecalciferol (Vit.D3 ) and 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma, Deisenhofen, Germany).

Cell growth and differentiation Cells were seeded at a density of 3 × 104 to 1 × 105 cells/ml and incubated in duplicate with various differentiation promoting agents for 96 h. Numbers of viable cells were determined daily by trypan blue exclusion using a haemocytometer. Phagocytic activity was determined by counting the number of cells capable of ingesting latex beads (0.81 mm diameter; Sigma, Deisenhofen, Germany) as described.52

temperature of 50°C. PCR-products were then analysed in 2% agarose gels.

Preparation of protein extracts Nuclear and cytosolic protein extracts were prepared in parallel according to published procedures.56 The cells were disrupted by homogenization in a dounce homogenizer and nuclei were sedimented by centrifugation. Cytosolic proteins were obtained from the supernatant after an additional clearance spin (14 000 × g for 10 min at 4°C). Extraction buffers were supplemented with 0.5 mM phenylmethylsulfonyl fluoride (Sigma, Deisenhofen, Germany) and 100 kallikrein inhibitory (KI) units/ml of aprotinin (Roth Chemicals, Karlsruhe, Germany). Whole cell extracts were prepared as described.50

RNA extraction and Northern blot analysis Electrophoretic mobility shift assays

From 2 × 108 cells total cellular RNA was isolated by the guanidine isothiocyanate caesium chloride method53 and polyA+-RNA was enriched by affinity chromatography on oligo-dT cellulose (Sigma, Deisenhofen, Germany). mRNAs were separated electrophoretically in agarose formaldehyde gels, blotted on Hybond N-filters (Amersham, Braunschweig, Germany) and fixed by UV-crosslinking. Prehybridization was performed in 50% formamide/5 × SSC (750 mM sodium chloride/75 mM sodium citrate)/1% SDS/ 2 × Denhardt’s (40 mg/ml each of polyvinylpyrrolidone, Ficoll 400 and bovine serum albumin) at 42°C. Filters were hybridized overnight under identical conditions with 50 ng of the appropriate DNA probe. Probes were labelled with [a-32P]dCTP (Amersham, Braunschweig, Germany) to specific activities of 2–6 × 108 cpm/mg using a commercial kit (Pharmacia, Freiburg, Germany). The following probes were used: A 1.8-kb BamHI fragment of the cDNA for mouse gp130 (provided by S. Akira, Osaka), a 0.6-kb HindIII fragment of the cDNA for the mouse IL-6-receptor (from G. Ciliberto, Rome) and PCR-generated fragments of the cDNA for murine Stat1 (nucleotides 1835 to 2228), Stat3 (nucleotides 479 to 997) and Stat5a (nucleotides 1192 to 1717). The cDNA fragments were cloned and verified by DNA sequence analysis. Filters were evaluated for constant loading by hybridization with a radiolabelled 0.8-kb PstI cDNA fragment for rat glyceraldehyde-3phosphate dehydrogenase (GAPDH). Sizes of mRNAs were determined by comparison of the specific signals with a RNA ladder (Gibco, Eggenstein, Germany). Intensities of autoradiographic signals were analysed by two-dimensional densitometry.

Protein–DNA-binding reactions were carried out for 20 min at room temperature in a 20-ml reaction volume containing 10 mM N-[2-Hydroxyethyl]piperazine-N'[2-ethanesulfonic acid] (HEPES), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 6 mM MgCl2 , 1.2 mM CaCl2 , 2 KI units of aprotinin, 2.5 mg of poly(dI:dC) (Pharmacia, Freiburg, Germany), 2.5 mg of single-stranded salmon sperm DNA, 10% glycerol, variable amounts of nuclear protein extract and approximately 7 fmol of the double-stranded, radiolabelled DNA probe TB2. This oligonucleotide contains two copies of the core-element of the IL-6-/LIF-response element (IL-6-/LIF-RE) from the rat a2-macroglobulin gene (TB2: 5'-GATCATCCTTCTGGGAATTCGATATC CTTCTGGGAATTCTG-3'; 42-mer; core-elements underlined).37,38 The IL-6-LIF-RE represents a Stat-binding site.27,38,39 TB2 was end-labelled with polynucleotide kinase and used at a specific activity of 3 × 106 cpm/pMol, corresponding to 2 × 104 cpm per reaction. EMSAcompetition analysis was performed with double-stranded oligonucleotides representing the Stat-binding site.37,56 For EMSA-supershift assays nuclear extracts were preincubated with Stat-specific antibodies (final dilution 1:20) for 1 h at 4°C. Thereafter, binding reactions and analysis of protein–DNA complexes were carried out as described.16,39 Anti-Stat1a and anti-Stat5a/MGF30 antibodies were provided by Drs J. E. Darnell Jr (New York) and B. Groner (Freiburg), respectively. Rabbit anti-Stat3 (C-20) antibodies and appropriate control antibodies were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

RT-PCR analysis

Immunoblot analysis

Residues 1677–1691 and 2174–2188 from the published cDNA sequence for the murine LIF-R54 were used for the sense and antisense primers, respectively. cDNA was prepared using 2 mg of total RNA and oligo-dT primers according to published procedures.55 PCR reactions were performed in a 50 ml volume containing 20 pmol of primers, 200 mmol of each deoxynucleotide triphosphate, and 2.5 units of Taq DNA polymerase (Perkin Elmer, Weiterstadt, Germany) in a final concentration of 10 mM Tris (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2 . Reaction mixtures were subjected to 35 successive PCR cycles with an annealing

Proteins were separated by polyacrylamide gel electrophoresis using denaturing conditions and transferred to nitrocellulose membranes (Schleicher & Schu¨ll, Dassel, Germany) by semi-dry blotting.55 Specific proteins were immunodetected using anti-Jak2 antibodies (provided by Dr J. Ihle, Memphis) and anti-Stat1a/b (M-22), anti-Stat3 (C-20) and anti-Stat5a (L-20) antibodies (Santa Cruz Biotechnology Inc., Heidelberg, Germany). Signals were visualized using peroxidase-conjugated secondary antibodies and an enhanced chemiluminescent system (Amersham, Braunschweig, Germany).

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Acknowledgements We are grateful to Drs B. Sola (Caen, France) and M. Kamps (San Diego, CA) for providing myeloid leukaemia cell lines. The authors wish to thank Drs J. Ihle (Memphis, TN), J. E. Darnell Jr (New York, NY) and B. Groner (Freiburg, Germany) for providing anti-Jak2, anti-Stat1a and anti-Stat5a/MGF antibodies, respectively. We thank Brigitte Go¨pfert for expert technical assistance and Dr G. Fey for helpful comments. This work was supported by research grants of the Deutsche Forschungsgemeinschaft (DFG; grant Hi 291/5-4 TP6) and the Sander Stiftung awarded to G.M.H. R.P.P. was the recipient of a graduate student fellowship from the Graduiertenkolleg ‘‘RNASynthese’’ supported by the DFG.

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