SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling

The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.     

Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colorectal cancer cells via multiple signaling pathways

Overexpression of cyclooxygenase-2 (COX-2) has been observed in human colorectal cancer. COX-2 expression in human tumors can be induced by growth factors, cytokines, oncogenes, and other factors. The mechanisms regulating COX-2 expression in human colon cancer have not been completely elucidated. We hypothesized that the proinflammatory cytokine interleukin-1 beta (IL-1 beta) mediates COX-2 expression in HT-29 human colon cancer cells. Treatment of HT-29 cells with IL-1 beta induced expression of COX-2 mRNA and protein in a time- and dose-dependent manner. Inhibitors of the extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, P38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-kappa B) signaling pathways blocked the ability of IL-1 beta to induce COX-2 mRNA. In contrast, Wortmannin, a phosphoinositide 3-kinase inhibitor upstream of protein kinase B/Akt, led to a slight increase in COX-2 mRNA expression after IL-1 beta treatment. Electrophoretic mobility shift assay on nuclear extracts demonstrated that IL-1 beta induced NF-kappa B DNA binding activity in HT-29 cells, and the activated NF-kappa B complex was eliminated after treatment with an inhibitor of NF-kappa B. Supershift assay indicated that the two NF-kappa B subunits, p65 and p50, were involved in activation of NF-kappa B complex by IL-1 beta stimulation. The stability of COX-2 mRNA was not altered by IL-1 beta treatment. These data demonstrate that IL-1 beta induces COX-2 expression in HT-29 cells through multiple signaling pathways and NF-kappa B.     

Insulin-like growth factor-1 induces adhesion and migration in human multiple myeloma cells via activation of beta1-integrin and phosphatidylinositol 3'-kinase/AKT signaling

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.     

Role of alpha 2 beta 1- and alpha 3 beta 1-integrin in the peritoneal implantation of scirrhous gastric carcinoma.

We established a highly peritoneal-seeding cell line, OCUM-2MD3, from a poorly peritoneal-seeding cell line, OCUM-2M, of human scirrhous gastric carcinoma. The intraperitoneal inoculation of OCUM-2MD3 cells produced peritoneal dissemination in nude mice, whereas that of OCUM-2M cells did not. We then investigated the correlation between seeding potential and adhesion molecule beta 1-integrins or alpha 6 beta 4-integrin. alpha 2 beta 1- and alpha 3 beta 1-integrin expression on OCUM-2MD3 cells (91.6% and 93.6%) was increased compared with that of OCUM-2M cells (47.8% and 34.3%) by flow cytometric analysis, and the expression level of the other integrins was not different between the two cell lines. The binding ability of OCUM-2MD3 cells to matrigel, fibronectin, laminin and type I collagen was significantly increased, approximately seven times, three times, eight times, and three times greater than that of OCUM-2M cells respectively. The invasiveness of OCUM-2MD3 cells was also significantly increased 8-fold over OCUM-2M cells. The binding and invasive ability of OCUM-2MD3 cells was significantly decreased following the addition of anti-alpha 2 beta 1- and alpha 3 beta 1-integrin antibody, but not by anti-alpha 6 beta 1- and alpha 6 beta 4-integrin antibody. These results suggest that adhesiveness and invasiveness in peritoneal implantation of scirrhous gastric carcinoma might be closely associated with alpha 2 beta 1- and alpha 3 beta 1-integrin.     

Simvastatin inactivates beta1-integrin and extracellular signal-related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cells

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell-extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of beta1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosis-inducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells.     

DF3/MUC1 signaling in multiple myeloma cells is regulated by interleukin-7

The human DF3/MUC1 transmembrane protein is aberrantly expressed in multiple myeloma cells and other B cell malignancies. The regulation of MUC1 in B cells and its potential function as a signaling molecule are unknown. The present results demonstrate that interleukin-7 (IL-7) stimulates MUC1 expression in multiple myeloma cells. The results also demonstrate the IL-7 induces binding of MUC1 to the Lyn tyrosine kinase. The MUC1 C-terminal subunit binds directly to Lyn through interactions with the Lyn SH3 and SH2 domains. Activation of Lyn in response to IL-7 stimulation results in increased tyrosine phosphorylation of the MUC1 C-terminal subunit. In vitro and in vivo studies show that Lyn phosphorylates MUC1, at least in large part, on a YEKV site in the MUC1 cytoplasmic tail. The functional significance of the MUC1-Lyn interaction is supported by the demonstration that Lyn-mediated phosphorylation of MUC1 on YEKV induces binding of MUC1 and the beta-catenin signaling protein. In concert with these results, IL-7 treatment is associated with binding of MUC1 to beta-catenin and targeting of the MUC1-beta-catenin complex to the nucleus. These findings indicate that IL-7 regulates MUC1 expression and function in multiple myeloma cells.     

Cyclin D1 induction through IkappaB kinase beta/nuclear factor-kappaB pathway is responsible for arsenite-induced increased cell cycle G1-S phase transition in human keratinocytes

Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G(1)-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-kappaB (NF-kappaB) activation, because the inhibition of IkappaB phosphorylation by overexpression of the dominant-negative mutant, IKKbeta-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IkappaB kinase beta (IKKbeta) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKbeta knockout (IKKbeta(-/-)) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1-specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G(1) to S phase transition is through IKKbeta/NF-kappaB/cyclin D1-dependent pathway.     

Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3beta (GSK-3beta) in vitro

Cell adhesion to extracellular matrix (ECM) is thought to confer resistance against cell-damaging agents, that is, drugs and radiation, in tumour and normal cells in vitro. The dependence of cell survival on beta1-integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt) and glycogen synthase kinase-3beta (GSK-3beta) activity, which participate in beta1-integrin signalling and cell cycle progression was investigated as a function of radiation exposure. Colony-formation assays on polystyrene, fibronectin (FN), laminin (LA), bovine serum albumin (BSA) or poly-L-lysine (poly-L) (0-8 Gy), kinase assays, flow cytometric DNA and annexin-V analysis and immunoblotting were performed in nonirradiated and irradiated (2 or 6 Gy) A549 human lung cancer cells and CCD32 normal human lung fibroblasts. Cell contact to FN in contrast to polystyrene elevated basal ILK, PKBalpha/Akt and GSK-3beta kinase activities in A549 and CCD32 cells, as well as the basal amount of A549 G2 phase cells. Irradiation on FN or LA as compared to polystyrene, BSA or poly-L significantly improved cell survival. Following irradiation, kinase activities were stimulated strongly on polystyrene but showed to be less prominent on FN, which was because of the FN-related basal induction. Following irradiation, FN compared to polystyrene enlarged and prolonged G2 arrest in both the cell lines. For the analysis of phosphatidylinositol-3 kinase (PI3-K) dependence of protein kinases and cell cycle transition, the PI3-K inhibitors LY294002 and wortmannin were used showing decreased kinase activities, antiproliferative and radiation-dependent G2 accumulation-abrogating effects accompanied by downregulation of cyclin D1 and phospho-pRb in cells attached to polystyrene. Fibronectin partly abrogated these effects PI3-K-independently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3beta by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBalpha/Akt and GSK-3beta are involved in modulations of the cell cycle after irradiation. These interactions are strictly dependent on ECM components in a cell line-specific manner. Our findings provide molecular insights into mechanisms likely to be important for ECM-dependent cell survival and cellular radioresistance as well as tumour growth.     

A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells

HER2 overexpression, a known prognostic factor in many human cancers, can activate phosphatidylinositol-3 kinase (PI-3K)/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia-inducible factors (HIFs) promote angiogenesis and energy metabolism and thereby enhance tumor growth and metastasis. HIFs, composed of alpha and beta subunits, are activated in most human cancers, including those that overexpress HER2. Previous reports have suggested that increased PI-3K/Akt or decreased PTEN activity may activate the HIF pathway in various tumors, but the detailed mechanism is still not completely understood. Here we reported an interaction between the HIF and PI-3K/Akt pathways in HER2-overexpressing cancer cells. Our results indicate that HER2 overexpression, which results in constitutively active Akt, turns on HIF-1alpha independently of hypoxia, and this activation is weaker than that under hypoxic condition. Further investigation showed that Akt is required for the hypoxia-independent HIF activity. The PI-3K/Akt pathway did not affect the HIF-1alpha binding with its E3 ligase von Hippel-Lindau but enhanced the binding affinity between the HIF-1 alpha and beta subunits. Furthermore, we found that Akt interacts with HIF-1beta and regulates HIF activity. Our results indicated that HER2 can induce HIF activation via the activation of Akt suggesting that activation of HER2/Akt pathway may promote angiogenesis independent of hypoxia, which may have important implications for the oncogenic activity of HER2 and Akt.     

Phosphorylation of BRCA2 by the Polo-like kinase Plk1 is regulated by DNA damage and mitotic progression

The breast cancer susceptibility protein, BRCA2, preserves chromosomal stability through roles in the repair of DNA double-strand breaks, and possibly, cell division. Post-translational modifications that may coordinate these functions remain poorly characterized. Here, we report that BRCA2 is a substrate for the mitotic Polo-like kinase, Plk1. BRCA2 undergoes phosphorylation in cells synchronously passing through the G2/M phases of cell cycle, when Plk1 expression and activity are maximal. Depletion of Plk1 by RNA interference suppresses BRCA2 modification. BRCA2 and Plk1 interact with one another in cell lysates, through a conserved region in BRCA2, which spans the eight BRC repeat motifs essential for its function in DNA repair. Within this region, residues positioned between BRC repeats--but not the repeat motifs themselves--are phosphorylated by Plk1. Interestingly, Plk1-mediated modification of BRCA2 during the G2/M phases is inhibited by treatment with the radiomimetic agent, adriamycin. Thus, our findings define a regulatory circuit for BRCA2 phosphorylation by Plk1 that is responsive to DNA damage as well as mitotic progression.     

H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase

It has been suggested that phosphorylation of the histone variant H2AX after ultraviolet light (UV) irradiation is triggered by DNA double-strand breaks induced as replication forks collide with UV-induced bulky lesions. More recently, it has been shown that UV-induced H2AX phosphorylation can also occur outside of S-phase, but the mechanism for this replication-independent induction is not well understood. In this study, we show that H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is induced in all phases of the cell cycle. Accumulation of DNA repair intermediates by inhibition of DNA repair synthesis resulted in a marked increase of H2AX phosphorylation in repair proficient but not repair-deficient xeroderma pigmentosum-A cells. Using chemical inhibitors of the PI(3)-like kinase family of protein kinases as well as ataxia telangiectasia mutated and Rad-3 related (ATR)-deficient Seckel syndrome cells and ataxia telangiectasia mutated-deficient ataxia telangiectasia cells, we show that the H2AX phosphorylation induced by accumulation of repair intermediates is mediated primarily by the ATR kinase. We suggest a model for UV light-induced phosphorylation of H2AX where in addition to replication blockage, DNA repair intermediates trigger H2AX phosphorylation via the ATR kinase.     

G3BP is overexpressed in human tumors and promotes S phase entry

The expression of the human Ras-GTPase activating protein (GAP)-binding protein (G3BP) was studied in human tumors and cell lines of different origins. Northern blot analysis and immunoblotting experiments showed enhanced expression of G3BP in all tumor samples as compared to healthy tissue. The enhanced expression does not seem to be related to the tumor site or to the stage of development of the cancer. In light of the proposed functions of G3BP, its increased expression in tumors suggest that it plays a role in dedifferentiation and proliferation processes. We also show that G3BP promotes S phase entry in cultured fibroblasts deprived of serum and that this function is dependent on the presence of the RNA binding domain of the protein.     

Inhibition of HER2-integrin signaling by Cucurbitacin B leads to in vitro and in vivo breast tumor growth suppression

HER2, an oncogenic receptor is overexpressed in about 25-30% of breast cancer patients. HER2 has been shown to play role in tumor promotion by having cross-talk with multiple oncogenic pathways in cancer cells. Our results show that Cucurbitacin B (CuB), a triterpenoid steroidal compound inhibited the growth of various breast cancer cells with an IC50 ranging from 18-50nM after 48 and 72 h of treatment. Our study also revealed the significant inhibitory effects of CuB on HER2 and integrin signaling in breast cancer. Notably, CuB inhibited ITGA6 and ITGB4 (integrin α6 & integrin β4), which are overexpressed in breast cancer. Furthermore, CuB also induced the expression of major ITGB1and ITGB3, which are known to cause integrin-mediated cell death. In addition, we observed that TGFβ treatment resulted in the increased association of HER2 with ITGA6 and this association was inhibited by CuB treatment. Efficacy of CuB was tested in vivo using two different orthotopic models of breast cancer. MDA-MB-231 and 4T-1 cells were injected orthotopically in the mammary fat pad of female athymic nude mice or BALB/c mice respectively. Our results showed that CuB administration inhibited MDA-MB-231 orthotopic tumors by 55%, and 4T-1 tumors by 40%. The 4T-1 cells represent stage IV breast cancer and form very aggressive tumors. CuB mediated breast tumor growth suppression was associated with the inhibition of HER2/integrin signaling. Our results suggest novel targets of CuB in breast cancer in vitro and in vivo.     

CHK1和CHK2 shRNA转染对食管癌细胞照射后G2期阻滞的影响

目的 观察RNA干扰细胞周期检测点激酶CHK1和CHK2表达对食管癌细胞照射后G2期阻滞的影响。方法 CHK1和CHK2基因各选择4段序列分别设计合成短发卡状RNA（shRNA）,分别与pENTR^TM／U6质粒连接后转染Eca109食管癌细胞。采用Western blotting检测CHK1和CHK2蛋白表达,RT-PCR检测其mRNA表达,流式细胞仪检测5Cy照射后细胞周期变化,克隆法检测5Cy照射后细胞存活率。结果 CHK1和CHK2基因各成功建立4个序列的shRNA连接质粒,转染Eca109细胞后均使其蛋白表达降低。用抑制效果最好的CHKI和CHK2shRNA转染Eca109细胞后,其mRNA表达下降;在转染后72h,子一代细胞CHK1和CHK2蛋白仍明显降低,并使5Cy照射后1h的CHK2-T68磷酸化水平降低,而对CHK1-S345磷酸化水平无明显影响。CHK1 shRNA转染的Ecal09细胞在5Gy照射后12h时,明显减轻G2期阻滞程度;转染后72h和5Cy照射后12h,子一代Eca109细胞G2期阻滞仍明显低于单纯5Gy照射组;而CHK2shRNA转染不影响照射后C2期阻滞。CHK1和CHK2shRNA转染可降低5Gy照射后Eca109细胞存活率。结论 shRNA转染对CHK1和CHK2蛋白表达的抑制效应至少可以持续3d,且可传递给子代细胞;CHK1 shRNA转染可以减轻Eca109细胞照射后G2期阻滞,增加放射敏感性。     

Inhibition of extracellular-signal regulated kinases 1/2 is required for apoptosis of human colon cancer cells in vitro by sulindac metabolites

Nonsteroidal anti-inflammatory drugs (NSAIDs) including sulindac have shown potent chemopreventive and tumor regressive effects against colorectal cancer, the second leading cause of cancer death in the United States. However, the mechanisms by which sulindac inhibits tumor cell growth are not completely understood. We previously reported that sulindac metabolites inhibit the mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascade in colorectal cancer cell lines at doses that induce apoptosis, and inhibition of MEK/ERK activity with U0126 is sufficient to induce apoptotic cell death. To determine whether inhibition of MEK/ERK activity is necessary for sulindac-induced apoptosis of human colon cancer cells, stable transfectants were created that express an activated MEK1 gene in HT29 cells. HT29-MEK1(R4F) clones displayed a 10- to 20-fold increase in MEK1 activity compared with control HT29-pCEP4 clones. When compared with control HT29-pCEP4 clones, HT29-MEK1(R4F) clones were resistant to both apoptosis and inhibition of ERK1/2 phosphorylation induced by sulindac metabolites. These results suggest that inhibition of MEK/ERK signaling is necessary for the induction of apoptosis by sulindac metabolites.     

ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells.

Overexpression of the growth factor receptor ErbB-2/Her2/Neu has been implicated in the development of non-small-cell lung cancer. We have reported that the transformation of human lung epithelial cells by c-erbB-2 also requires an active ErbB-1 (EGF receptor) and the autocrine production of its ligand, TGF-alpha. In this report, we demonstrate that STAT 3 is constitutively activated in these cells by the TGF-alpha-stimulated ErbB-1/-2 heterodimer complex. STAT 3 activation was confirmed by mobility shift assays and nuclear localization. ErbB-1 was required, but not sufficient for the TGF-alpha-induced activation of STATs. Inhibition of ErbB-2 kinase activity by tyrphostin AG825 prevented the constitutive activation of STAT 3 in the TGF-alpha-producing, ErbB-1 expressing cell line. Our results demonstrate a requirement for ErbB-2 kinase activity to establish constitutive STAT 3 activation resulting from an autocrine ErbB-1/ TGF-alpha loop. Int. J. Cancer 83:564-570, 1999. Published 1999 Wiley-Liss, Inc.     

Transforming growth factor-beta 1-induced apoptosis is blocked by beta 1-integrin-mediated mitogen-activated protein kinase activation in human hepatoma cells

Growth factors and extracellular matrices cooperatively regulate cellular behavior. However, the interactions between transforming growth factor-beta 1 (TGF-beta 1) and integrins in hepatic cells are not fully understood. We investigated the effects of beta 1-integrin on TGF-beta 1-regulated growth of hepatoma cells. Human hepatoma cell lines HepG2, Huh7, and Hep3B were stably transfected with beta 1-integrin, and the parental, and mock- and beta 1-integrin-transfected hepatoma cells were treated with TGF-beta 1. Modulation of apoptosis and pathways involved in the process were investigated. TGF-beta 1 suppressed the growth of hepatoma cells, and apoptosis was observed in Hep3B and Huh7. Hepatoma cells transfected with beta 1-integrin were protected from TGF-beta 1-induced apoptosis. Mitogen-activated protein (MAP) kinase inhibitors, PD98059, SB203580, and SP600125, abolished this protective effect of beta 1-integrin, but herbimycin A and wortmannin were ineffective. Hepatoma cells overexpressing beta 1-integrin showed increased activities of MAP kinases, and TGF-beta 1 induced sustained activation of MAP kinases in these cells, but only transient activation in mock-transfected cells. These data suggest that MAP kinases activated by beta 1-integrin provide a strong anti-apoptotic signal during TGF-beta 1-induced apoptosis in human hepatoma cells. Therefore beta 1-integrin-mediated signals may contribute to the development and progression of hepatocellular carcinoma.