Interleukin-11 expression: its significance in eutopic and ectopic human implantation.

Embryo implantation and subsequent decidualization, trophoblast invasion and formation of a functional placenta are crucial for establishment and maintenance of pregnancy. Interleukin-11 signalling has been shown to be obligatory for adequate decidualization and trophoblast invasion in mice. Defects in IL-11 signalling in mice result in trophoblast over-invasion and fetal loss. The pathological situation of human tubal pregnancy resembles that of IL-11Ralpha(-/-) mice concerning these symptoms. As our interest is focused on the human early pregnancy, we compared IL-11 expression at the implantation site of ectopic tubal pregnancy (EP) to 1st and 2nd trimester of normal intrauterine pregnancies (IP), and to the normal cycling endometrium. The mRNA expression of IL-11 and IL-11Ralpha was analysed by semiquantitative RT-PCR. Protein expression was detected by western blotting and immunohistochemistry. IL-11Ralpha is expressed constitutively in all tissue specimens analysed. IL-11 is expressed predominantly during follicular and early luteal phase of the menstrual cycle. In IP, IL-11 expression peaks during the 1st trimester and declines from the beginning of the 2nd trimester onwards. In tubal abortions, IL-11 expression is reduced in comparison to vital EP and IP. Cultured primary endometrial and decidual epithelial cells were analysed for hormonal regulation of IL-11 by enzyme-linked immunosorbent assay and RT-PCR. IL-11 is up-regulated by estrogen and down-regulated by progesterone. Overall, our results indicate that in humans, IL-11 signalling is significantly involved in regulation of trophoblast invasion. In the case of tubal abortion, inadequate IL-11 signalling may therefore result in dysregulation of trophoblast invasion.

The cytokine receptor gp130 and its soluble form are under hormonal control in human endometrium and decidua.

The transmembrane protein gp130 plays a central role in cytokine action as a signal transducing receptor subunit common to all interleukin-6 type cytokines. Endometrial tissue obtained from women with a normal menstrual cycle and decidua obtained from women in the first or second trimester of pregnancy were assessed for gp130 by western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis. By immunoblotting, two forms of gp130 were detected: one-the soluble form-of approximately 100 kDa and a larger membrane-bound form of approximately 150 kDa. The latter became clearly visible in the mid to late secretory phase and was more pronounced in decidual tissue of second trimester compared to first trimester. Immunohistochemically, gp130 was located in glandular epithelial cells during the mid to late secretory phase, whereas staining in the proliferative phase was rather weak. In first and second trimester decidua, glandular cells were also positively stained. In addition, the invading trophoblast cells were gp130 positive. Soluble gp130 release was measured in the supernatants from primary endometrial and decidual cell cultures by ELISA and reached maximum values in cell cultures without addition of hormones. In cultured endometrial epithelial cells obtained during the proliferative phase of the cycle, the soluble gp130 release increased significantly under combined estradiol/progesterone supplementation which mimics the secretory phase conditions compared to estradiol supplementation alone. In cultured epithelial cells derived from decidual tissue of first trimester of pregnancy, similar effects of hormonal regulation were observed. Our results suggest that the balance between soluble gp130 and its membrane-bound form may play an important role in regulating cytokine action necessary for blastocyst implantation and for further interaction between the decidualized endometrium and the invading trophoblast.

The MKK6/p38 mitogen-activated protein kinase pathway is capable of inducing SOCS3 gene expression and inhibits IL-6-induced transcription.

In this study we show that activation of p38MAPK by IL-6 acts as an inhibitory signal on IL-6-mediated activation of STAT and the alpha2-macroglobulin promoter. We analyzed the role of MKK6/p38MAPK for IL-6 signal transduction and transcriptional activation of the suppressor of cytokine signaling (SOCS) 3 promoter. Pretreatment of cells with the p38MAPK-specific inhibitor SB202190 downregulates the induction of SOCS3-mRNA expression by IL-6. Accordingly, overexpression of a constitutively active MKK6 in HepG2 cells enhanced basal activity or IL-6-induced transcriptional activation of a SOCS3 promoter reporter construct, whereas overexpression of a dominant negative mutant of MKK6 downregulated the IL-6-mediated activation of the SOCS3 promoter. These data indicate that p38MAPK-activation is crucial for IL-6-induced SOCS3 expression and downregulation of IL-6-mediated gene induction.

Interleukin-6-induced proliferation of pre-B cells mediated by receptor complexes lacking the SHP2/SOCS3 recruitment sites revisited.

Interleukin-6 (IL-6) induces B-cell proliferation by binding to receptor complexes composed of a specific alpha-receptor (gp80; CD126) and the signal transducing receptor subunit gp130 (CD130). Immediately after receptor complex activation, signal transducers and activators of transcription (STATs) 1 and 3 and the Src-homology domain-containing protein tyrosine phosphatase 2 (SHP2) are recruited to gp130 and subsequently tyrosine phosphorylated. The activated dimerized STATs translocate to the nucleus and bind to enhancer elements of IL-6-inducible genes. SHP2 acts as an adapter and links the Jak/STAT pathway to the Ras/Raf/MAPK cascade but it is also involved in signal attenuation. Whereas STAT3 activation appears to be crucial for all biological activities of IL-6, the requirement of SHP2-activation depends on the individual biological response analyzed. The requirement of SHP2 activation for the pre-B cell (Ba/F3) proliferation has been reported previously [Fukada, T., Hibi, M., Yamanaka, Y., Takahashi-Tezuka, M., Fujitani, Y., Yamaguchi, T., Nakajima, K. & Hirano, T. (1996) Immunity 5, 449-460]. In contrast, we have recently demonstrated that the presence of a single STAT-recruitment site within gp130 is sufficient for IL-6- induced proliferation of Ba/F3 cells [Schmitz, J., Dahmen, H., Grimm, C., Gendo, C., Müller-Newen, G., Heinrich, P.C. & Schaper, F. (2000) J. Immunol. 164, 848-854]. To unravel this discrepancy we analyzed the IL-6-induced dose-dependent proliferation of Ba/F3 cells mediated by receptor complexes lacking SHP2/SOCS3 recruitment sites. Surprisingly, pre-B cells, after stimulation with low amounts of IL-6, proliferate much more efficiently in the absence of the activated SHP2 than in the presence of the tyrosine phosphatase. Therefore, SHP2 activation appears to be relevant for IL-6-induced proliferation only after stimulation with very large amounts of IL-6.

The vesicular stomatitis virus matrix protein inhibits glycoprotein 130-dependent STAT activation.

Infection of cells by vesicular stomatitis virus (VSV) results in the inhibition of host transcription. We show in this study that infection of HeLa cells with VSV leads to a strongly diminished activation of STAT3 and STAT1 by the inflammatory cytokine IL-6. This effect was mimicked by forced expression of a single viral protein, the matrix (M)-protein of VSV, which blocked STAT activation via chimeric receptors containing the cytoplasmic domain of the IL-6 signal transducer gp130. Western blot analysis revealed that VSV M-protein did not inhibit the nuclear translocation of activated STAT3 but did inhibit its tyrosine phosphorylation. Inhibition of STAT activation was not dependent on tyrosine 759 of the IL-6 signal transducer gp130, suggesting that the inhibitory action of VSV M-protein is not mediated by the induction of the suppressor of cytokine signaling 3. VSV M-protein inhibited gene transcription from cotransfected alpha(2)-macroglobulin or antichymotrypsin promoter/luciferase reporter constructs which contain STAT3-binding sites. However, transcription from a STAT5-dependent construct was not negatively affected. In conclusion, our data suggest that infection by VSV and specifically overexpression of the viral M-protein interferes with an important signaling pathway necessary for triggering antiviral and inflammatory responses.

A completely foreign receptor can mediate an interferon-gamma-like response.

A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.

The inhibitory effect of IL-1 beta on IL-6-induced alpha 2-macroglobulin expression is due to activation of NF-kappa B.

The cross-talk between the signal transduction of simultaneous acting cytokines largely determines the final impact of cytokines on their target genes. Both NF-kappaB and STAT3 are transcription factors well known to be activated by many stimuli and to mediate transcriptional activation by binding to specific enhancer sequences. In this study, it is analyzed how IL-1beta inhibits IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. It is shown that IL-1beta prevents STAT3 binding to the two STAT3-responsive sites within the alpha(2)-macroglobulin promoter by association of IL-1beta-activated NF-kappaB to this region. The observation that inhibition of IL-6-induced transcriptional activation of this promoter by IL-1beta is reversed by cotransfection with I-kappaBalpha provides evidence that NF-kappaB activation by IL-1beta is responsible for inhibition of IL-6-mediated trans activation of the alpha(2)-macroglobulin gene. Accordingly, cotransfection of the NF-kappaB subunits p50 or p65 themselves inhibited activation of the alpha(2)-macroglobulin promoter by IL-6. Introduction of point mutations in each of the two NF-kappaB sites overlapping the two STAT3 binding sites within the alpha(2)-macroglobulin promoter provides evidence that each of these two sites counteracts transcriptional activation via STAT3. Most interestingly, at least one functional NF-kappaB consensus site is essential for the IL-6-induced transcriptional activation of the alpha(2)-macroglobulin promoter. Additional data are provided indicating that the activation of NF-kappaB by IL-1beta is also responsible for the inhibition of other IL-6-inducible genes, such as the alpha(1)-antichymotrypsin gene as well as the suppressor of cytokine signaling 3 gene, suggesting a more general relevance of this mechanism for transcriptional regulation.

Interleukin-6-resistant melanoma cells exhibit reduced activation of STAT3 and lack of inhibition of cyclin E-associated kinase activity.

Development of cytokine resistance is an important feature of melanoma cells during tumor progression. To study the mechanisms of interleukin-6 resistance, we examined an interleukin-6 sensitive (WM35) and an interleukin-6 unresponsive cell line (WM9). Interleukin-6 treatment resulted in rapid inhibition of cyclin-dependent kinase 2/cyclin E activity and accumulation of the hypophosphorylated retinoblastoma protein in WM35 but not in WM9 cells. In contrast to previous reports, no differences in the expression of the cyclin-dependent kinase 2 inhibitor p21Cip1/WAF1 upon interleukin-6 treatment were found in both cell lines. Interleukin-6-induced inhibition of cyclin-dependent kinase 2 was also not due to changes in protein expression of cyclin-dependent kinase 2, cyclin E, p27Kip1 and cdc25A, a phosphatase positively regulating cyclin-dependent kinase 2 activity. As it is established that interleukin-6 resistance of WM9 cells is not caused by differential interleukin-6 receptor expression, we studied whether this is due to defective interleukin-6 signaling in which activation of signal transducer and activator of transcription 3 is a critical step. WM9 cells showed reduced tyrosine phosphorylation, DNA binding, and delayed nuclear translocation of signal transducer and activator of transcription 3 as compared with WM35 cells. The kinase upstream of signal transducer and activator of transcription 3, Janus kinase 1, was constitutively tyrosine-phosphorylated in WM9 cells and did not respond to interleukin-6 with increased phosphorylation. As compared with WM35 cells, interleukin-6 treatment of WM9 cells was not paralleled by reduced activity of the mitogen-activated protein kinase kinase-1, which suppresses activation of signal transducer and activator of transcription 3. Our data suggest that resistance of advanced melanoma cells to interleukin-6 is associated with reduced inhibition of cyclin-dependent kinase 2, which appears to be a consequence of a complex alteration in interleukin-6 signal transduction.

STAT3 is constitutively activated in Hodgkin cell lines.

Hodgkin disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg cells are rare and surrounded by abundant reactive nonmalignant cells. It has been suggested that cytokines such as interleukin-6 (IL-6) are involved in the pathogenesis of the disease. The expression of the IL-6 receptor (IL-6R) complex and its link to the activation of signal transducers and activators of transcription (STAT) molecules in HD cell lines was investigated. Gel retardation and Western blot analyses revealed a high level of constitutively activated STAT3 in 5 of 7 HD cell lines, which could not be detected in Burkitt lymphoma cell lines. Different levels of IL-6R protein were measured in various HD cell lines: L428 and Dev cells were characterized by very low levels of gp80 and gp130, on KMH2 cells only gp130 but no gp80 was detected, whereas L540, L591, HDLM2, and L1236 were positive for both gp80 and gp130, suggesting a possible autocrine stimulation of STAT3. However, a further increase in STAT3 activation on IL-6 or IL-6/soluble IL-6R stimulation was not observed. Neutralizing monoclonal antibodies against IL-6, gp80, gp130, or both receptor subunits did not affect the proliferation or the constitutive activation of STAT molecules in HD cell lines. However, the tyrosine kinase inhibitor AG490 blocked the constitutive activation of STAT3 and inhibited spontaneous growth of HD tumor cells. The evidence suggests abnormal STAT signaling and growth regulation in Hodgkin cell lines. (Blood. 2001;98:762-770)

Mapping of a region within the N terminus of Jak1 involved in cytokine receptor interaction.

Janus kinase 1 (Jak1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors. Here we show that the in vitro translated N-terminal domains of Jak1 are sufficient for binding to a biotinylated peptide comprising the membrane-proximal 73 amino acids of gp130, the signal-transducing receptor chain of interleukin-6-type cytokines. By the fold recognition approach amino acid residues 36-112 of Jak1 were predicted to adopt a beta-grasp fold, and a structural model was built using ubiquitin as a template. Substitution of Tyr(107) to alanine, a residue conserved among Jaks and involved in hydrophobic core interactions of the proposed beta-grasp domain, abrogated binding of full-length Jak1 to gp130 in COS-7 transfectants. By further mutagenesis we identified the loop 4 region of the Jak1 beta-grasp domain as essential for gp130 association and gp130-mediated signal transduction. In Jak1-deficient U4C cells reconstituted with the loop 4 Jak1 mutants L80A/Y81A and Delta(Tyr(81)-Ser(84)), the interferon-gamma, interferon-alpha, and interleukin-6 responses were similarly impaired. Thus, loop 4 of the beta-grasp domain plays a role in the association of Jak1 with both class I and II cytokine receptors. Taken together the structural model and the mutagenesis data provide further insight into the interaction of Janus kinases with cytokine receptors.

Mitogen-activated protein kinases control p27/Kip1 expression and growth of human melanoma cells.

The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated protein kinase (ERK)1 and ERK2, involved in regulating cell growth and differentiation, are constitutively active in A375 and WM239 human melanoma cells. Using PD098059, an inhibitor of MAPK kinase (MEK), we investigated the role of persistently activated ERK1/2 in cell growth. The inhibition of MAPK activity induced a dose-dependent growth arrest in G(0)/G(1) phase. Correspondingly, we observed the up-regulation of the cyclin-dependent kinase (Cdk) inhibitor p27/Kip1 and hypophosphorylation of the retinoblastoma protein. Further studies showed that PD098059 treatment significantly decreased Cdk2 kinase activity, most probably owing to an augmented level of p27/Kip1 associated with cyclin E-Cdk2 complexes. The accumulation of p27/Kip1 protein in A375 cells was attributed to its increased stability. Our findings suggest that constitutively active ERK1/2 kinases contribute to the growth of melanoma cells by negative regulation of the p27/Kip1 inhibitor.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

Identification of the domain in the human interleukin-11 receptor that mediates ligand binding.

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.

Two different epitopes of the signal transducer gp130 sequentially cooperate on IL-6-induced receptor activation.

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.

Non-redundant signal transduction of interleukin-6-type cytokines. The adapter protein Shc is specifically recruited to rhe oncostatin M receptor.

The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr(861) of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr(861) is essential for activation of ERK1/2 and for full activation of the alpha(2)-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.

The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized madin-darby canine kidney cells.

The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain.

In vitro-generated stem cell leukaemia showing altered cell cycle progression with distinct signalling of the tyrosine-phosphorylated rasGAP-associated p62(dok) protein.

In an attempt to gain more insight into the events of leukaemic transformation, a cell line overexpressing MHC class II (DR) was generated by transfecting an early CD34-negative haematopoietic progenitor stem cell line with the appropriate constructs. The stable transfection with genes for DR antigens leads to cellular transformation. The DR(+) transformed cell clones express a tyrosine-phosphorylated DR heterodimer and show a significantly different morphology. DR(+) clones present the morphology of an immature myeloid neoplasia expressing alpha-naphthyl-acetate-esterase (ANAE), but neither myeloperoxidase nor CD34. While D064 cells predominately grow adherent as fibroblast-like cells, the DR(+) clones display a decrease in adherent growth. Although both cell lines express similar amounts of the interleukin-6 (IL-6) signal transducer gp130, the DR-transfected cells still show activation of STAT factors by IL-6, whereas D064 cells do not. Although the transformed clones present acceleration of cell-cycle transition and growth, the G(0)/G(1) progression inhibitor p27(kip-1) is up-regulated, while the expression of proteins involved in the S/G(2) phase transition, such as cyclin B and cdc2 (p34), is suppressed. Instead cyclin D3, one of the G(0)/G(1) progression factors, is up-regulated, as well as tyrosine-phosphorylated p62(dok), suggesting dysregulation of cell cycle-controlling proteins. In addition, DR(+) leukaemia-like cells also overexpress Bcl-2, while bax expression is suppressed, compared with the wild-type (wt) parental haematopoietic stem cell line.