Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.     

All-trans-retinoic acid induces tyrosine phosphorylation of the CrkL adapter in acute promyelocytic leukemia cells

OBJECTIVE: All-trans-retinoic acid (RA) is a potent inducer of differentiation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. It also exhibits synergistic effects with interferons on the induction of differentiation and growth inhibition in vitro. Recent studies showed that interferons engage a signaling pathway involving the CBL proto-oncogene and the CrkL adapter, which mediates interferon-induced growth inhibitory signals. The objective of this study was to determine whether the CBL-CrkL pathway is activated by treatment of the NB-4 and HL-60 acute leukemia cell lines with RA. MATERIALS AND METHODS: The effects of RA treatment on CBL and CrkL phosphorylation, as well as on protein-protein interactions, were determined in studies involving immunoprecipitations of cell extracts with specific antibodies and Western blots. In addition, glutathione-S-transferase fusion proteins were used in binding studies to determine whether the SH2 domain of CrkL interacts with CBL in a RA-dependent manner and whether Rapl is activated by RA. RESULTS: Treatment of NB-4 or HL-60 cells with RA resulted in strong tyrosine phosphorylation of CBL, which was time and dose dependent. Similarly, RA induced tyrosine phosphorylation of the CrkL adapter and the association of CrkL with CBL. The RA-dependent interaction of CrkL with CBL was mediated by binding of the SH2 domain of CrkL to tyrosine phosphorylated CBL, suggesting that CBL provides a docking site for engagement of CrkL in a RA-activated cellular pathway. The guanine exchange factor C3G was found to be associated with CrkL at similar levels before and after RA treatment, but Rapl activation downstream of C3G was not inducible by RA. CONCLUSIONS: These findings demonstrate that the CBL-CrkL pathway is one of the mediators of the effects of RA on APL cells and suggest that one of the mechanisms of synergy between RA and interferons may involve regulation of components of this signaling cascade.     

The adapter protein CrkL associates with CD34

CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)- fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i(full)). Precipitation of cell lysates using GST-CD34i(full) identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GST-CrkL3' (C-terminal SH3) and GST-CrkL5' (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3' could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34, because GST-CD34i(full) did not precipitate CrkII, a highly homologous Crk family member. Furthermore, GST-CD34i(full) did not bind c-Abl, c-Cbl, C3G, or paxillin proteins that are known to associate with CrkL, suggesting that CD34 directly interacts with the CrkL protein. CD34i(full) association with Grb or Shc adapter proteins was not detected. Our investigations shed new light on signaling pathways of CD34 by demonstrating that CD34 couples to the hematopoietic adapter protein CrkL. (Blood. 2001;97:3768-3775)     

Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells

Stem cell factor (SCF) plays critical roles in proliferation, survival, migration, and function of hematopoietic progenitor and mast cells through binding to Kit receptor. Previous studies have implicated the adaptor protein Lnk as an important negative regulator of SCF signaling. However, the molecular mechanism underlying this regulation is unclear. Here, we showed that the Src homology 2 domain (SH2) of Lnk binds directly and preferentially to phosphorylated tyrosine 567 in Kit juxtamembrane domain. Using Lnk(-/-) bone marrow mast cells (BMMCs) transduced with different Lnk proteins, we demonstrated that Lnk down-regulates SCF-induced proliferation with attenuation of mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase signaling. Furthermore, we showed that Lnk(-/-) BMMCs displayed increased SCF-dependent migration compared with wild-type cells, revealing a novel Lnk-mediated inhibitory function. This correlated with enhanced Rac and p38 MAPK activation. Finally, we found that Lnk domains and carboxy-terminal tyrosine contribute differently to inhibition of in vitro expansion of hematopoietic progenitors. Altogether, our results demonstrate that Lnk, through its binding to Kit tyrosine 567, negatively modulates specific SCF-dependent signaling pathways involved in the proliferation and migration of primary hematopoietic cells.     

Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.     

Constitutive c-jun N-terminal kinase activity in acute myeloid leukemia derives from Flt3 and affects survival and proliferation

OBJECTIVE: c-jun N-terminal kinase (JNK) has been implicated in proliferation and survival downstream from the tyrosine kinase oncogene, p210 BCR-ABL, in chronic myeloid leukemia. We studied whether a similar relationship between JNK and FMS-like tyrosine kinase 3 (Flt3) describes acute myeloid leukemia (AML). METHODS: By immunoprecipitation, Flt3 was found to be activated and identified as the potential origin of JNK activity in a heavy majority of JNK+ve AML blasts tested. Often, Flt3 activity is associated with activating mutation of the gene locus. However, statistical linkage tied JNK activity with Flt3 expression levels rather than with mutation. An adaptor network to describe the signal cascade Flt3-to-JNK was uncovered. RESULTS: Active Flt3 was linked to p85 phosphoinositide-3 (PI-3) kinase, and p85 with cbl and CrkII/CrkL by co-immunoprecipitaton assays from lysates of model cell lines and primary AML blasts. JNK1 co-immunoprecipitated from such lysates with p85-cbl-crkII/L and bound to Crk species SH3 domain in pull-down assay. siRNA-mediated depletion of Flt3 or of cbl, the adaptor at the nexus of this signaling group, inhibited JNK activity on substrate c-jun. Within AML blast cells influenced by Flt3 signaling, selective inhibition of JNK by a small molecule inhibitor, led to proliferative inhibition, apoptosis, and sensitizing cells to the anthracycline, daunorubicin. These effects occurred upon JNK inhibition without off-target inhibition of extracellular signal-regulated kinase or AKT pathways, and p38-kinase activation, an effector in the p53/p14 arf tumor suppressor pathway, was also maintained or augmented. CONCLUSION: JNK is a bonafide signaling pathway from Flt3 in AML whose function for proliferation and survival is required in a significant AML cohort with active Flt3 signaling, by mutation or overexpression of Flt3.     

Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.     

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 × 10⁹/l. P2l0^BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p2l0^BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.     

Lupus-like disease and high interferon levels corresponding to trisomy of the type I interferon cluster on chromosome 9p

OBJECTIVE: Systemic lupus erythematosus (SLE) is associated with type I interferons (IFNs) and can be induced by IFNalpha treatment. This study looked for evidence of autoimmunity in a pedigree consisting of 4 family members with a balanced translocation 9;21 and 2 members with an unbalanced translocation resulting in trisomy of the short (p) arm and part of the long (q) arm of chromosome 9. These latter 2 subjects had 3 copies of the IFN gene cluster. METHODS: Subjects were evaluated clinically and serologically for autoimmune disease. Expression levels of IFNalpha4, IFNbeta, the type I IFN-inducible gene Mx1, the type I IFN receptor, interleukin-6, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction. Circulating plasmacytoid dendritic cells, the main IFN-producing cells, were quantified by flow cytometry. RESULTS: Both subjects with trisomy of chromosome 9p had a lupus-like syndrome with joint manifestations and antinuclear antibodies: one had anti-RNP and antiphospholipid autoantibodies, and the other had anti-Ro 60. The 3 family members with a balanced translocation 9;21 had no clinical or serologic evidence of autoimmunity, similar to that in relatives who were unaffected by the chromosomal translocation. In the 2 subjects with trisomy of 9p, high levels of IFNalpha/beta (comparable with those found in patients with SLE), increased signaling through the IFN receptor (as indicated by high Mx1 expression), and low levels of circulating plasmacytoid dendritic cells (as observed in patients with SLE) were evident. These abnormalities were not seen in individuals with a balanced translocation. CONCLUSION: Trisomy of the type I IFN cluster of chromosome 9p was associated with lupus-like autoimmunity and increased IFNalpha/beta and IFN receptor signaling. The data support the idea that abnormal regulation of type I IFN production is involved in the pathogenesis of SLE.     

Requirement of type I interferon signaling for arthritis triggered by double-stranded RNA

OBJECTIVE: Arthralgias and overt arthritides are often associated with viral infections. Viral infections expose the infected host to proinflammatory double-stranded RNA (dsRNA), which can cause joint inflammation and is a potent activator of interferon-alpha (IFNalpha). The aim of this study was to determine the role of IFNalpha and dsRNA-related signaling molecules in the onset of joint inflammation induced by viral dsRNA. METHODS: IFNalpha and different forms of RNA were injected into the knee joints of wild-type mice, mice lacking the type I interferon receptor (IFNAR(-/-)), and mice deficient in dsRNA-dependent protein kinase (PKR(-/-)). Histologic evidence of joint damage and the ability of splenocytes to produce cytokines in response to dsRNA or IFNalpha were assessed. RESULTS: Viral dsRNA, but not short single-stranded RNA, induced arthritis. The arthritis was aggravated by intracellular delivery of dsRNA. The expression of PKR was not mandatory for dsRNA-induced joint inflammation. In contrast, IFNalpha/beta signaling was important for dsRNA-induced joint inflammation because IFNAR(-/-) mice did not develop arthritis. Furthermore, intraarticular deposition of IFNalpha induced arthritis in PKR(-/-) and control mice, whereas IFNAR(-/-) mice were protected. The arthritogenic effect of IFNalpha was attenuated by in vivo depletion of monocyte/macrophages. CONCLUSION: Arthritis triggered by dsRNA is not dependent on the expression of the dsRNA-signaling molecule PKR (or Toll-like receptor 3, as previously shown), but is associated with the ability to produce type I IFN and is critically dependent on type I IFN receptor signaling. The intrinsic arthritogenic properties of IFNalpha implicate a role of this cytokine in joint manifestations triggered by various interferogenic stimuli.     

Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering

BACKGROUND & AIMS: The pathogenesis of Helicobacter pylori (Hp)-associated diseases depends on a specialized type IV secretion system. This type IV secretion system injects the cytotoxin-associated gene A (CagA) effector protein into target cells where CagA becomes phosphorylated on tyrosine residues by Src. Src then is inactivated rapidly, suggesting the presence of another host tyrosine kinase to ensure constant CagA phosphorylation in sustained Hp infections. We aimed to identify this kinase. METHODS: By using the AGS gastric epithelial cell model, we performed a detailed functional characterization of Abl tyrosine kinase in signaling during Hp infections. RESULTS: We showed that Abl kinase is activated and a novel crucial mediator of Hp infections. First, Abl-specific inhibitors SKI-DV2-43 or STI571 (Gleevec, Novartis) and knockdown of c-Abl/Abl-related gene Arg by small hairpin and interfering RNAs efficiently inhibit CagA phosphorylation and cell scattering. Second, during infection, Abl is activated rapidly by autophosphorylation at Y-412. Third, both Abl and Src phosphorylated Y-899, Y-918, and Y-972 of CagA. Fourth, we found that the Abl substrate CrkII is phosphorylated at Y-221 in vivo. Fifth, overexpression of kinase-dead Abl (K290M) blocked Hp-induced actin cytoskeletal rearrangements. We further showed that sustained activity of Abl is required to maintain CagA in a phosphorylated state. Moreover, phosphorylated CagA forms a physical complex with Abl and activated CrkII in vivo. CONCLUSIONS: We propose a model in which Hp has evolved a mechanism to use at least 2 tyrosine kinases, Abl and Src, for CagA phosphorylation and subsequent actin-cytoskeletal rearrangements leading to cell scattering and elongation.     

Integrin engagement-induced inhibition of human myelopoiesis is mediated by proline-rich tyrosine kinase 2 gene products

OBJECTIVE: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells. METHODS: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays. RESULTS: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors. CONCLUSION: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.     

Activation of the type I interferon system in primary Sjögren's syndrome: a possible etiopathogenic mechanism

OBJECTIVE: The etiopathogenesis of primary Sj?gren's syndrome (SS) is largely unknown. In other autoimmune diseases, type I interferon (IFN) may play a pivotal role by triggering and sustaining the disease process. We therefore aimed to determine whether patients with primary SS had an activated type I IFN system. METHODS: Salivary gland biopsy specimens and sera from patients with primary SS were investigated for the occurrence of IFNalpha-producing cells and measurable IFNalpha levels, respectively. The ability of primary SS sera together with apoptotic or necrotic cells to induce IFNalpha production in normal peripheral blood mononuclear cells was examined. The IFNalpha inducer was characterized, and IFNalpha-producing cells were identified. Clinical data were correlated with the IFNalpha-inducing capacity of primary SS sera. RESULTS: Numerous IFNalpha-producing cells were detected in salivary gland biopsy specimens, despite low serum IFNalpha levels. Autoantibodies to RNA-binding proteins, combined with material released by necrotic or late apoptotic cells, were potent inducers of IFNalpha production in plasmacytoid dendritic cells (PDCs). This appeared to be attributable to RNA-containing immune complexes triggering PDCs by means of RNA and interaction with Fcgamma receptor IIa. The IFNalpha-inducing capacity of sera was associated with positive results of a labial salivary gland biopsy (focus score >/=1) and with dermatologic, hematologic, and pulmonary manifestations. CONCLUSION: Patients with primary SS have an activated type I IFN system. Although virus may initiate the production of IFN, the continued IFNalpha synthesis is caused by RNA-containing immune complexes that activate PDCs to prolong IFNalpha production at the tissue level. This IFNalpha promotes the autoimmune process by a vicious circle-like mechanism, with increased autoantibody production and formation of more endogenous IFNalpha inducers.     

The Ebolavirus VP24 protein blocks phosphorylation of p38 mitogen-activated protein kinase

Type I interferon (IFN) signaling is mediated through several signaling pathways, including the Janus kinase and signal transducer and activator (JAK-STAT) and p38 mitogen-activated protein (MAP) kinase pathways. The VP24 protein of Ebolavirus is an IFN antagonist, blocking type I IFN signaling through the JAK-STAT pathway. Here, we show that, in 293 cells, VP24 also interferes with the p38 MAP kinase pathway by blocking IFN-β-stimulated phosphorylation of p38-α. Similar inhibition was not observed in HeLa cells, suggesting cell type-specific differences in signal transduction.     

DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10-related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.     

Potent inhibitory effects of type I interferons on human adrenocortical carcinoma cell growth

CONTEXT: Adrenocortical carcinoma (ACC) is a rare tumor with a poor prognosis. Despite efforts to develop new therapeutic regimens for metastatic ACC, surgery remains the mainstay of treatment. Interferons are known to exert tumor-suppressive effects in several types of human cancer. DESIGN: We evaluated the tumor-suppressive effects of type I interferons (IFN)-alpha2b and IFNbeta on the H295 and SW13 human ACC cell lines. RESULTS: As determined by quantitative RT-PCR analysis and immunocytochemistry, H295 and SW13 cells expressed the active type I IFN receptor (IFNAR) mRNA and protein (IFNAR-1 and IFNAR-2c subunits). Both IFNalpha2b and IFNbeta1a significantly inhibited ACC cell growth in a dose-dependent manner, but the effect of IFNbeta1a (IC50 5 IU/ml, maximal inhibition 96% in H295; IC50 18 IU/ml, maximal inhibition 85% in SW13) was significantly more potent, compared with that of IFNalpha2b (IC50 57 IU/ml, maximal inhibition 35% in H295; IC50 221 IU/ml, maximal inhibition 60% in SW13). Whereas in H295 cells both IFNs induced apoptosis and accumulation of the cells in S phase, the antitumor mechanism in SW13 cells involved cell cycle arrest only. Inhibitors of caspase-3, caspase-8, and caspase-9 counteracted the apoptosis-inducing effect by IFNbeta1a in H295 cells. In H295 cells, IFNbeta1a, but not IFNalpha2b, also strongly suppressed the IGF-II mRNA expression, an important growth factor and hallmark in ACC. CONCLUSIONS: IFNbeta1a is much more potent than IFNalpha2b to suppress ACC cell proliferation in vitro by induction of apoptosis and cell cycle arrest. Further studies are required to evaluate the potency of IFNbeta1a to inhibit tumor growth in vivo.