Targeting Src reactivates pyroptosis to reverse chemoresistance in lung and pancreatic cancer models.

Pancreatic and lung cancers frequently develop resistance to chemotherapy-induced cell apoptosis during the treatment, indicating that targeting nonapoptotic-related pathways, such as pyroptosis, can be an alternative cancer treatment strategy. Pyroptosis is a gasdermin-driven lytic programmed cell death triggered by inflammatory caspases when initiated by canonical or noncanonical pathways that has been recently seen as a potential therapeutic target in cancer treatment. However, overcoming chemoresistance in cancers by modulating pyroptosis has not been explored. Here, we demonstrate that ß5-integrin represses chemotherapy-induced canonical pyroptosis to confer cancer chemoresistance through ASAH2-driven sphingolipid metabolic reprogramming. Clinically, high ß5-integrin expression associates with poor patient prognosis and chemotherapeutic responses in cancers. In addition, chemoresistant cells in vitro fail to undergo chemotherapy-induced pyroptosis, which is controlled by ß5-integrin. Mechanistically, proteomic and lipidomic analyses indicate that ß5-integrin up-regulates sphingolipid metabolic enzyme ceramidase (ASAH2) expression through Src-signal transducer and activator of transcription 3 (STAT3) signaling, which then reduces the metabolite ceramide concentration and subsequent ROS production to prohibit chemotherapy-induced canonical pyroptosis. Using cancer cell lines, patient-derived tumor organoids, and orthotopic lung and pancreatic animal models, we show that administration of a Src or ceramidase inhibitor rescues the response of chemoresistant pancreatic and lung cancer cells to chemotherapy by reactivating pyroptosis in vitro and in vivo. Overall, our results suggest that pyroptosis-based therapy is a means to improve cancer treatment and warrants further investigation.

SOX2-mediated upregulation of CD24 promotes adaptive resistance toward targeted therapy in melanoma.

Melanoma is often characterized by a constitutively active RAS-RAF-MEK-ERK pathway. For targeted therapy, BRAF inhibitors are available that are powerful in the beginning but resistance occurs rather fast. A better understanding of the mechanisms of resistance is urgently needed to increase the success of the treatment. Here, we observed that SOX2 and CD24 are upregulated upon BRAF inhibitor treatment. A similar upregulation was seen in targeted therapy-resistant, melanoma-derived induced pluripotent cancer cells (iPCCs). SOX2 and CD24 are known to promote an undifferentiated and cancer stem cell-like phenotype associated with resistance. We, therefore, elucidated the role of SOX2 and CD24 in targeted therapy resistance in more detail. We found that the upregulation of SOX2 and CD24 required activation of STAT3 and that SOX2 induced the expression of CD24 by binding to its promoter. We find that the overexpression of SOX2 or CD24 significantly increases the resistance toward BRAF inhibitors, while SOX2 knock-down rendered cells more sensitivity toward treatment. The overexpression of CD24 or SOX2 induced Src and STAT3 activity. Importantly, by either CD24 knock-down or Src/STAT3 inhibition in resistant SOX2-overexpressing cells, the sensitivity toward BRAF inhibitors was re-established. Hence, we suggest a novel mechanism of adaptive resistance whereby BRAF inhibition is circumvented via the activation of STAT3, SOX2 and CD24. Thus, to prevent adaptive resistance, it might be beneficial to combine Src/STAT3 inhibitors together with MAPK pathway inhibitors.

GLP-1 inhibits VEGFA-mediated signaling in isolated human endothelial cells and VEGFA-induced dilation of rat mesenteric arteries.

We investigated the acute effects of glucagon-like peptide-1 (GLP-1), GLP-1(1-36), and GLP-1(7-36) on vascular endothelial growth factor-A (VEGFA)-induced endothelium-dependent signaling and vasodilation. Our hypothesis was that GLP-1 released from intestinal l-cells modulates processes related to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries. In isolated rat mesenteric resistance arteries precontracted with the thromboxane analog U46619 to 80-90% of maximum contraction, VEGFA (25 ng/ml) caused a small and gradual relaxation (28.9 ± 3.9%). Pretreatment of arteries with either GLP-1(1-36) (500 nM) or GLP-1(7-36) (1 nM) abolished the VEGFA-induced relaxation. VEGFA-induced relaxations were also inhibited in endothelial-denuded arteries and in arteries pretreated with the nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester (100 µM). In vivo studies on male Wistar rats also revealed that GLP-1(7-36) inhibited VEGFA-induced vasodilation of the same arteries. In isolated endothelial cells, GLP-1(1-36) and GLP-1(7-36) caused a reduction in VEGFA-induced phosphorylation of PLCγ. Ca2+ imaging of endothelial cells and rat mesenteric resistance arteries using fura-2, revealed that both GLP-1 analogs caused a reduction in VEGFA-induced Ca2+ signaling. GLP-1(1-36) also reduced VEGFA-induced eNOS phosphorylation in HDMEC. In conclusion, GLP-1 reduced relaxation induced by VEGFA in resistance arteries by inhibiting VEGFR2-mediated Ca2+ signaling and endothelial NO synthesis. GLP-1, on its own, also induced phosphorylation of Src and ERK1/2 that can lead to proliferation and is implicated in vessel permeability.

CD14 as a Mediator of the Mineralocorticoid Receptor-Dependent Anti-apolipoprotein A-1 IgG Chronotropic Effect on Cardiomyocytes.

In vitro and animal studies point to autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) as possible mediators of cardiovascular (CV) disease involving several mechanisms such as basal heart rate interference mediated by a mineralocorticoid receptor-dependent L-type calcium channel activation, and a direct pro-inflammatory effect through the engagement of the toll-like receptor (TLR) 2/CD14 complex. Nevertheless, the possible implication of these receptors in the pro-arrhythmogenic effect of anti-apoA-1 antibodies remains elusive. We aimed at determining whether CD14 and TLRs could mediate the anti-apoA-1 IgG chronotropic response in neonatal rat ventricular cardiomyocytes (NRVC). Blocking CD14 suppressed anti-apoA-1 IgG binding to NRVC and the related positive chronotropic response. Anti-apoA-1 IgG alone induced the formation of a TLR2/TLR4/CD14 complex, followed by the phosphorylation of Src, whereas aldosterone alone promoted the phosphorylation of Akt by phosphatidylinositol 3-kinase (PI3K), without affecting the chronotropic response. In the presence of both aldosterone and anti-apoA-1 IgG, the localization of TLR2/TLR4/CD14 was increased in membrane lipid rafts, followed by PI3K and Src activation, leading to an L-type calcium channel-dependent positive chronotropic response. Pharmacological inhibition of the Src pathway led to the decrease of L-type calcium channel activity and abrogated the NRVC chronotropic response. Activation of CD14 seems to be a key regulator of the mineralocorticoid receptor-dependent anti-apoA-1 IgG positive chronotropic effect on NRVCs, involving relocation of the CD14/TLR2/TLR4 complex into lipid rafts followed by PI3K and Src-dependent L-type calcium channel activation.

PPemd26, an anthraquinone derivative, suppresses angiogenesis via inhibiting VEGFR2 signalling.

BACKGROUND AND PURPOSE: Angiogenesis contributes to coronary heart disease, immune disorders and numerous malignancies. VEGF-A and its receptors (VEGFRs) play a pivotal role in regulating angiogenesis. In an effort to discover more effective inhibitors of tumour angiogenesis, we have analysed the actions of a novel anthraquinone derivative, PPemd26, and explored its anti-angiogenic mechanisms. EXPERIMENTAL APPROACH: The effects of PPemd26 were evaluated in vitro using HUVEC cultures to assess proliferation, migration, invasion and tube formation. Immunoblotting was used to analyse phosphorylation of signalling kinases. Effects in vivo were assayed using Matrigel plug and xenograft mouse models. KEY RESULTS: PPemd26 significantly inhibited VEGF-A-induced proliferation, migration, invasion and tube formation of HUVECs. PPemd26 also attenuated VEGF-A-induced microvessel sprouting from rat aortic rings ex vivo and suppressed formation of new blood vessels in implanted Matrigel plugs in models of angiogenesis in vivo. In addition, PPemd26 inhibited VEGF-A-induced phosphorylation of VEGFR2 and its downstream protein kinases including Akt, focal adhesion kinase, ERK and Src. Furthermore, systemic administration of PPemd26 suppressed the growth of s.c. xenografts of human colon carcinoma in vivo. Histochemical analysis of the xenografts revealed a marked reduction in stainingfor the vascular marker CD31 and proliferation marker Ki-67. CONCLUSIONS AND IMPLICATIONS: This study provides evidence that PPemd26 suppressed tumour angiogenesis through inhibiting VEGFR2 signalling pathways, suggesting that PPemd26 is a potential drug candidate for developing anti-angiogenic agents for the treatment of cancer and angiogenesis-related diseases.

Lysophosphatidic acid rescues human dental pulp cells from ischemia-induced apoptosis.

INTRODUCTION: Dental pulp is particularly susceptible to ischemic conditions (hypoxia and serum deprived) because it is commonly exposed to trauma, inflammation, chronic caries injury, and pulpitis. We investigated the apoptotic response of human dental pulp cells (HDPCs) to varying levels of oxygen and serum to mimic different degrees of ischemia, tested whether lysophosphatidic acid (LPA) could reverse ischemia-induced apoptosis, and investigated the possible mechanisms of LPA. METHODS: HDPCs were cultured under conditions mimicking serum deprivation and ischemia for 2 days with or without LPA at 25 µg/mL. Flow cytometry and JC-1 fluorescence were used to detect any apoptotic change. Western blotting was used to measure the expression of the apoptosis regulators B-cell lymphoma 2 (Bcl-2) and Bax, focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK), and Akt. RESULTS: Flow cytometry and JC-1 immunofluorescence showed that ischemia could induce apoptosis of HDPCs in 2 days and treatment with LPA could reduce cell death significantly. To clarify the molecular mechanisms, Western blot results showed up-regulation of both proapoptotic Bax and antiapoptotic Bcl-2 during apoptosis. LPA functioned as an antiapoptotic cytokine by activation of the phosphorylation of FAK and ERK. No statistically significant difference was found in the activation levels of p-Src or p-Akt. CONCLUSIONS: A self-defense mechanism functioned during cell apoptosis. LPA could effectively rescue HDPCs from ischemia-induced apoptosis via regulation of Bax and Bcl-2 and the activation of phosphorylated FAK and phosphorylated ERK. LPA is a potent candidate for biological therapy of chronic pulpal inflammatory diseases.

Concomitant targeting of EGF receptor, TGF-beta and SRC points to a novel therapeutic approach in pancreatic cancer.

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-ß) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-ß sequestration by soluble TGF-ß receptor II (sTßRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTßRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTßRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTßRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTßRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTßRII due to increased cancer cell proliferation. The combination of shEGFR and sTßRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTßRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-ß, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-ß-dependent pathways.

New molecular targets in bone metastases.

Bone metastases have a major impact on morbidity and on mortality in cancer patients. Despite its clinical relevance, metastasis remains the most poorly elucidated aspect of carcinogenesis. The biological mechanisms leading to bone metastasis establishment have been referred as "vicious circle," a complex network between cancer cells and the bone microenvironment. This review is aimed to underline the new molecular targets in bone metastases management other than bisphosphonates. Different pathways or molecules such as RANK/RANKL/OPG, cathepsin K, endothelin-1, Wnt/DKK1, Src have recently emerged as potential targets and nowadays preclinical and clinical trials are underway. The results from those in the advanced clinical phases are encouraging and underlined the need to design large randomised clinical trials to validate these results in the next future. Targeting the bone by preventing skeletal related events (SREs) and bone metastases has major clinical impact in improving survival in bone metastatic patients and in preventing disease relapse in adjuvant setting.

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel.

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.

Involvement of EGF receptor and c-Src in the survival signals induced by TGF-beta1 in hepatocytes.

Transforming growth factor beta1 (TGF-beta1) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. Although solid data established the role of TGF-beta's as suppressor factors in tumorigenic processes, in the context of an advanced stage of disease, TGF-beta's could also play a pro-oncogenic role. We have previously shown that TGF-beta1 induces both pro- and anti-apoptotic signals in foetal rat hepatocytes. In this work, we have focused on its anti-apoptotic mechanism. We show that TGF-beta1 activates the epidermal growth factor receptor (EGFR) and phosphorylates c-Src. EGFR is required for Akt activation. Blocking EGFR signalling amplifies the apoptotic response to TGF-beta1. TGF-beta1 induced a rapid activation of the tumour necrosis factor-alpha-converting enzyme (TACE/ADAM (a disintegrin and metalloprotease) 17). Inhibitors of TACE considerably attenuated Akt activation, which suggests that TGF-beta1 activates EGF signalling in hepatocytes by promoting shedding of EGF-like ligands. The activation of c-Src by TGF-beta1 is EGFR dependent and is required for full Akt phosphorylation and cell survival. Inhibition of EGFR does not block the epithelial-mesenchymal transition (EMT) induced by TGF-beta1 in hepatocytes, which indicates that activation of EGFR plays an essential role in impairing apoptosis, but it is dispensable for the EMT process.

Extracellular pressure stimulates colon cancer cell adhesion in vitro and to surgical wounds by Src (sarcoma protein) activation.

BACKGROUND: We hypothesized that pressure stimulates colon cancer cell adhesion to surgical wounds. METHODS: We quantitated adhesion of murine 26/51 transplantable colon cancer cells by cell counting or chromium 51-labeling. Tumor cells were added to murine surgical wounds after 30 minutes preincubation under ambient or 15 mm Hg increased pressure. Src activation was assayed by immunoblotting for phosphorylated Src and inhibited by 4-amino-5-(4chlorophenyl)-7-(t-butyl)pyrazolo-[3-4-d]pyrimidine (PP2). RESULTS: Pressure stimulated colon 26/51 cell adhesion to murine wounds by 43% to 52% (n = 9, P <0.05 each). Pressure stimulated Src phosphorylation by 39% +/- 4% (n = 5, P = 0.004) in colon 26 cells. The Src inhibitor PP2 (20 mumol/L) did not inhibit Src phosphorylation at ambient pressure but prevented pressure stimulation of Src phosphorylation. Src blockade by PP2 did not affect basal adhesion of either tumor to murine wounds but completely blocked pressure stimulation of adhesion (n = 4, P <0.001 each). CONCLUSIONS: Increased pressure may activate cancer adhesion to surgical wounds via Src. Src antagonists might inhibit this process.

c-Src protein tyrosine kinase activity is required for muscarinic receptor-mediated DNA synthesis and neurogenesis via ERK1/2 and c-AMP-responsive element-binding protein signaling in neural precursor cells.

The G protein-coupled muscarinic acetylcholine receptor (mAChR) isoforms have been identified in neural stem/progenitor (or precursor) cells. In previous studies, activation of these receptors induced elevations in intracellular Ca(2+) signals and mitogen-activated protein (MAP) kinase activity that led to enhanced DNA synthesis along with neurogenesis in neural precursor cells. Here we report that the nonreceptor protein tyrosine kinase c-src activity is required for the muscarinic receptor-activated MAP kinase and cAMP-responsive element-binding protein (CREB). Stimulation of neural precursor cells dissociated from embryonic day 13 rat cortical neuroepithelium with the muscarinic receptor agonist carbachol (CCh) induced phosphorylations of c-src that were detected by antibodies raised against phospho-Tyr416 (Ptyr416), phospho-Tyr527 (Ptyr527), and phospho-Tyr215 (Ptyr215) of the kinase. Although an increase in Ptyr416 suggested direct activation of c-src, Ptyr215 may serve as an alternative mechanism underlying activation of c-src without dephosphorylation of Ptyr-527. Both extracellular signal-regulated kinase (Erk1/2) and CREB were significantly activated after CCh treatment indicated by increases in phosphorylation of these two proteins. The c-Src inhibitor PP1 abolished the CCh-induced activation of Erk1/2 and CREB in a dose-dependent manner. Moreover, CCh stimulated expression of the neuronal specific marker MAP2, which was inhibited by PP1. Cell proliferation assays and immunocytochemistry revealed that PP1 inhibited the CCh-induced DNA synthesis and MAP2(+) production. These results suggest that c-src activity is essential for the muscarinic receptor-mediated proliferation and neurogenesis in neural precursor cells via Erk1/2 and CREB signaling pathways.

Attenuation of pressure-induced myogenic contraction and tyrosine phosphorylation by fasudil, a cerebral vasodilator, in rat cerebral artery.

The mechanism by which fasudil inhibits pressure-induced myogenic contraction was studied with regard to tyrosine phosphorylation in rat cerebral artery. Intracellular Ca(2+) concentration ([Ca(2+)](i)) and vessel diameter were simultaneously measured. Total tyrosine phosphorylation level and phosphorylation of tyrosine 419 on pp60(src) required for its full catalytic activity were immunocytochemically detected in situ. Fasudil (1 - 100 microM) partially suppressed the increase in [Ca(2+)](i), and totally attenuated contraction elicited by pressurization from 10 to 60 mmHg. Furthermore, fasudil (100 microM) significantly attenuated tyrosine phosphorylation and the activity of pp60(src) augmented in situ by pressure. Herbimycin A (1 - 100 nM) and genistein (3 - 30 microM), tyrosine kinase inhibitors, effectively attenuated the pressure-induced increase in [Ca(2+)](i), contraction, tyrosine phosphorylation, and activation of pp60(src). Both fasudil and herbimycin A directly inhibited the pp60(src) activity in a cell free system. Orthovanadate (100 microM), a tyrosine phosphatase inhibitor, significantly potentiated the pressure-induced increase in [Ca(2+)](i) and contraction. Nicardipine (100 nM), a Ca(2+) antagonist, completely inhibited pressure-induced increase in [Ca(2+)](i) and contraction, but affected neither tyrosine phosphorylation nor activity of pp60(src) in the pressurized arteries. Arginine-glycine-aspartic acid-serine peptide (1 - 100 microM) concentration-dependently reduced the pressure-induced contraction. In addition to the hitherto reported vasodilatory actions of fasudil, the present results suggest the inhibition by fasudil of pressure-induced tyrosine phosphorylation and pp60(src) activation. The wide spectrum of inhibitory actions of fasudil may contribute to the effective attenuation of the pressure-induced contraction in the cerebral artery.

Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity.

1. Tyrosine kinases have been proposed as regulators of voltage-operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I(Ba)) and pp60(c-src) kinase (c-src) activity in vitro. 2. I(Ba) was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage-clamp techniques. The activity of purified human c-src was measured in vitro using a non-radioactive assay. 3. Bath application of tyrphostin-23 and genistein (non-selective PTK inhibitors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src family-selective inhibitor) inhibited I(Ba) in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of peptide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor potency series against I(Ba) was PP1 > genistein > tyrphostin 23 > bistyrphostin. 4. Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on I(Ba) activation calculated from current-voltage relationships. 5. The agents inhibited c-src activity in a concentration-dependent manner. The order of potency was PP1 > genistein > peptide-A > tyrphostin-23 > bistyrphostin. The IC(50) for inhibition of c-src activity was similar to the IC(50) for inhibition of I(Ba) in all cases. 6. Western blot analysis with a specific antibody to c-src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7. A range of structurally dissimilar inhibitors of PTKs inhibit I(Ba) and c-src activity with similar potency. These data provide further evidence implicating endogenous c-src in the modulation of L-type calcium channels in vascular smooth muscle cells.

Opposing effects of cyclosporin A and tyrphostin AG-1478 indicate a role for Src protein in the cellular control of mineralization.

Cyclosporin A (CsA) induces osteoporosis but not through direct activation of osteoclasts. CsA also inhibits cell-mediated mineralization in marrow stromal cell culture, whereas the tyrphostin AG-1478 increases mineralization. These antagonistic effects on mineralization were used to discern molecules that underwent phosphorylation changes in association with their opposing effects on mineralization. In parallel, quantitative changes in Src protein were followed. Multiple dexamethasone (DEX)-stimulated stromal cell cultures were grown with and without a mineralization-inhibiting dose (0.1 microM) of CsA and were harvested on different days of DEX stimulation. Immunoblots of gel-fractionated cell extracts showed that the most noticeable changes in tyrosine phosphorylated proteins (TPP) were seen on day 8 of DEX stimulation. At least 15 TPP bands, mostly smaller than 53 kDa, were more prominent in CsA-treated cultures on day 8. Under CsA, Src protein quantity decreased on day 8, but its cleavage product (52/54 kDa) was sixfold more abundant then on day 7. Day 8 was chosen to test the effect of AG-1478 on the CsA-induced TPP changes. Dimethyl sulfoxide (DMSO) alone, the solvent of AG-1478, increased mineralization in CsA-treated versus CsA-untreated cultures and slightly decreased Src and its cleavage product. AG-1478 at 5 microM, in CsA cultures increased the specific alkaline phosphatase activity threefold, with a slight change in mineralization relative to controls grown with DMSO alone. This was accompanied by decreased intensity of several TPP bands smaller than 36 kDa. In contrast, treatment with 50 microM of AG-1478 increased the intensity of TPP bands at the same molecular size range. This high AG-1478 dose decreased cell counts selecting mineralizing cells. The results indicate that increased Src protein cleavage product on day 8 by CsA is associated with mineralization inhibition, which is opposed by DMSO and 50-microM AG-1478, thus antagonizing the effect of CsA on mineralization. Direct or indirect interaction between Src and TPP, antagonistically affected by CsA and AG-1478, is likely to underlay cellular control of mineralization. Changes in p19 and p29 intensity showed association with mineralization that was reflected by a significant direct and inverse correlation, respectively, with calcium precipitation per cell.

Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain.

Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor.

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells.

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.

Correcting temperature-sensitive protein folding defects.

Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein. Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy. Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected. These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities. We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases.

Mycotrienins. A new class of potent inhibitors of osteoclastic bone resorption.

Pharmacological intervention using selective tyrosine kinase inhibitors has been shown to be an effective approach to inhibit osteoclast function. Here, we report on the structure-activity relations of benzoquinone ansamycins isolated from Streptomyces rishirensis, which form a new class of potent inhibitors of osteoclast-mediated bone resorption. Parathyroid hormone-stimulated bone resorption was inhibited concentration dependently by both mycotrienin I and mycotrienin II, showing half-maximal inhibition in the low nanomolar range in fetal rat long bones in vitro. Structure-activity relation studies indicate that position 19 contained within the quinone/hydroquinone element and the double bonds in position 4, 6, and 8 are crucial for full bioactivity. In contrast, substitutions in position 22 are well tolerated. The lack of a similar effect of 2,6-dimethyl-p-benzoquinone and vitamin K signifies that the mechanism of action is not solely due to the oxygen scavenger capacity of the quinone/hydroquinone moiety. The inhibition of osteoclastic bone resorption is in line with the diminished activity of immunopurified pp60c-src from bone suggesting that pp60c-src is a possible target of mycotrienins in the organ culture. Thus, mycotrienins may be useful as pharmacologic inhibitors of osteoclastic bone resorption.