Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells.

Elevated focal adhesion kinase (FAK) expression in human tumor cells has been correlated with an increased cell invasion potential. In cell culture, studies with FAK-null fibroblasts have shown that FAK function is required for cell migration. To determine the role of elevated FAK expression in facilitating epidermal growth factor (EGF)-stimulated human adenocarcinoma (A549) cell motility, antisense oligonucleotides were used to reduce FAK protein expression >75%. Treatment of A549 cells with FAK antisense (ISIS 15421) but not a mismatched control (ISIS 17636) oligonucleotide resulted in reduced EGF-stimulated p130(Cas)-Src complex formation, c-Jun NH(2)-terminal kinase (JNK) activation, directed cell motility, and serum-stimulated cell invasion through Matrigel. Because residual FAK protein in ISIS 15421-treated A549 cells was highly phosphorylated at the Tyr-397/Src homology (SH)2 binding site, expression of the FAK COOH-terminal domain (FRNK) was also used as an inhibitor of FAK function. Adenoviral-mediated infection and expression of FRNK promoted FAK dephosphorylation at Tyr-397, resulted in reduced EGF-stimulated JNK as well as extracellular-regulated kinase 2 (ERK2) kinase activation, inhibited matrix metalloproteinase-9 (MMP-9) secretion, and potently blocked both random and EGF-stimulated A549 cell motility. Equivalent expression of a FRNK (S-1034) point-mutant that did not promote FAK dephosphorylation also did not affect EGF-stimulated signaling or cell motility. Dose-dependent reduction in EGF-stimulated A549 motility was observed with the PD98059 MEK1 inhibitor and the batimastat (BB-94) inhibitor of MMP activity, but not with the SB203580 inhibitor of p38 kinase. Finally, comparisons between normal, FAK-null, and FAK-reconstituted fibroblasts revealed that FAK enhanced EGF-stimulated JNK and ERK2 kinase activation that was required for cell motility. These data indicate that FAK functions as an important signaling platform to coordinate EGF-stimulated cell migration in human tumor cells and support a role for inhibitors of FAK expression or activity in the control of neoplastic cell invasion.

Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase.

Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Inhibition of c-Jun N-terminal kinase 1, but not c-Jun N-terminal kinase 2, suppresses apoptosis induced by ischemia/reoxygenation in rat cardiac myocytes.

In the present study, rat cardiac myocytes were used as an in vitro ischemia/reperfusion injury model to delineate the role of c-Jun N-terminal kinase (JNK) 1 and JNK2 isoforms in ischemia/reoxygenation-induced apoptosis using an antisense approach. Exposure of rat cardiac myocytes to ischemia did not induce apoptosis as detected by staining with either acridine orange/ethidium bromide or annexin-V-fluorescein/propidium iodide. In contrast, a time-dependent increase in the number of apoptotic cells was noted after reoxygenation of ischemic myocytes, whereas the level of necrotic cells remained unaltered. Reoxygenation, but not ischemia alone, also caused a time-dependent increase in JNK activation that preceded apoptosis induction. Treatment of cardiac myocytes with antisense (AS) oligonucleotides that specifically targeted either JNK1 or JNK2 significantly reduced both mRNA and protein expression of the target isoform but had no effect on the expression of the alternate isoform. Pretreatment of cardiac myocytes with JNK1 AS, but not JNK2 AS, resulted in almost complete attenuation of reoxygenation-induced apoptosis. Furthermore, control oligonucleotides for JNK1 AS or JNK2 AS had no effect on JNK mRNA or protein expression or reoxygenation-induced apoptosis, indicating a sequence-specific mode of action. Additional studies revealed that apoptosis induced by other JNK-activating stimuli, including ceramide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS but not JNK2 AS. These findings demonstrate that the JNK1 isoform plays a preferential role in apoptosis induced by ischemia/reoxygenation as well as diverse JNK-activating cellular stresses.

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade.

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.

c-Jun N-terminal kinase is essential for growth of human T98G glioblastoma cells.

The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway is activated by numerous cellular stresses. Although it has been implicated in mediating apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here, the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth-inhibitory potency of JNK2 antisense ((JNK)2 IC(50) = 0.14 micrometer) was greater than that of JNK1 antisense ((JNK)1 IC(50) = 0.37 micrometer), suggesting that JNK2 plays a dominant role in regulating growth of T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) accompanied by inhibition of Cdk2/Cdc2 kinase activities. Taken together, these results indicate that JNK is required for growth of T98G cells in nonstress conditions and that p21(cip1/waf1) may contribute to the sustained growth arrest of JNK2-depleted T98G cultures.

Elucidating cell signaling mechanisms using antisense technology.

Many diseases result from defects in cell signaling. Achieving an in-depth understanding of the complex mechanisms by which cells transduce extracellular signals into cellular responses in both normal and diseased systems is a crucial step in the discovery of more effective drugs to treat human diseases. Traditional approaches for studying cell signaling have some limitations. Antisense oligonucleotides represent a novel approach for studying signal transduction processes that offers significant advantages in terms of specificity and versatility. This article reviews the opportunities that antisense oligonucleotides offer for the study of signal transduction pathways and identification of inhibitors of these pathways for drug development.

Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells.

17-fold) increase in DNA fragmentation. Fluorescence microscopy, using DNA binding dyes, demonstrated that cell death following hypoxia/reoxygenation was due predominantly to apoptosis and not necrosis. Furthermore, reoxygenation, but not hypoxia alone, caused a time-dependent increase in the activation of JNK as monitored by western blot analysis using a phospho-specific JNK antibody. In contrast, p38 mitogen-activated protein kinase was activated following hypoxia, but this activation was not augmented during reoxygenation. Exposure of human kidney cells to a 2'-methoxyethyl mixed backbone antisense oligonucleotide directed against human JNK1 (JNK1 AS) resulted in a potent suppression of JNK mRNA and protein expression, whereas a 6-base mismatch control oligonucleotide was without effect. Moreover, a significant diminution of reoxygenation-induced apoptosis was observed in cells exposed to JNK1 AS but not to the mismatch control oligonucleotide. Taken together, these results strongly indicate that activation of the JNK signaling cascade is a major mechanism whereby hypoxia/reoxygenation induces apoptosis.

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner.

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.

Distinct roles of JNKs/p38 MAP kinase and ERKs in apoptosis and survival of HCD-57 cells induced by withdrawal or addition of erythropoietin.

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis.

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Interleukin-6 serum levels correlate with footpad swelling in adjuvant-induced arthritic Lewis rats treated with cyclosporin A or indomethacin.

To investigate the role of interleukin 6 (IL-6) in adjuvant-induced arthritis, serum from adjuvant-immunized Lewis rats treated with cyclosporin, indomethacin, or saline was evaluated for IL-6 activity. Inflammation was quantitated by measuring paw volume. We found that an increase in serum IL-6 activity parallels the kinetics of paw edema development in adjuvant-immunized rats. Daily treatment with 5 mg cyclosporin A/kg prevented the increase in paw volume and held serum IL-6 activity to levels observed in untreated (normal) rats. Daily treatment with 1 mg indomethacin/kg resulted in a 50% reduction in serum IL-6 levels and a significant decrease (approximately 50%) in paw volume on Day 17 compared to saline-treated rats. Linear regression analysis confirmed the positive correlation between mean paw volume and mean serum IL-6 activity (R2 = 0.783, P less than 0.01 on Day 17) in normal, arthritic, and cyclosporin A- or indomethacin-treated groups. These results are consistent with a role for IL-6 in the pathology of arthritis and suggest that serum IL-6 activity may be a useful parameter for monitoring disease activity.

Parameters altering the success of the one-anastomosis rat heart-lung transplant.

The single-anastomosis heart-lung transplantation method in the rat developed by Lee and associates has been examined and has proven to be simple and reproducible. Technical procedures are discussed emphasizing those procedures that require special caution to ensure successful graft function. The donor and recipient rat strain combination has been found to influence the success of the transplantation procedure. Rat strain combinations used in our laboratories resulted in different percentages of donor heart survival. Using Lewis rat (RT1 1/1) recipients, 68% of Brown Norway rat (RT1n/n) donor hearts and 97% of NBR rat (RT1 1/1) donor hearts survived. However, only 29% of Fischer rat (RT1 1/1) donor hearts survived longer than 2 days when transplanted into Lewis rat recipients. In our laboratories, the Lee method of single-anastomosis heart-lung transplantation has been used successfully to evaluate whether immunoregulatory compounds alter heart allograft rejection.

Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

Triton X-100-insoluble residues, or skeletons, of plasma membrane-rich vesicles obtained from unstimulated human platelets were isolated by high speed centrifugation. About 10-15% of the total surface iodinatable glycoproteins IIb and III (GPIIb and GPIII, respectively) co-isolated with the insoluble fraction. After sonication and centrifugation the solubilized material was further purified by affinity chromatography on Lens culinaris lectin-Sepharose. SDS PAGE analysis of this material revealed the presence of at least three major proteins, which were shown to be GPIIb, GPIII, and membrane actin, as judged by their electrophoretic properties and on the basis of immunological criteria. Antibodies directed against platelet surface glycoproteins and antibodies directed against rabbit actin were able to immunoprecipitate all three proteins, which indicates that they were noncovalently associated with one another. Gel filtration of the Lens lectin-purified Triton-insoluble complex on Ultrogel AcA 22 showed that greater than 85% of the total surface GPIIb and III was associated with an actin-rich peak that eluted in the void volume. In contrast, the form of GPIIb-III present in the Triton-soluble membrane fraction behaved as monomeric species when chromatographed under identical conditions. Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPIIb and III exist: one that binds directly to endogenous membrane actin and one that does not.

Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies.

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.