Active targeting using HER-2-affibody-conjugated nanoparticles enabled sensitive and specific imaging of orthotopic HER-2 positive ovarian tumors.

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER-2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.

HER-2/neu targeted delivery of a nanoprobe enables dual photoacoustic and fluorescence tomography of ovarian cancer.

Development of sensitive and specific imaging approaches for the detection of ovarian cancer holds great promise for improving survival of ovarian cancer patients. Here we describe a dual-modality photoacoustic and fluorescence molecular tomography (PAT/FMT) approach in combination with a targeted imaging probe for three-dimensional imaging of ovarian tumors in mice. We found that the selective accumulation of the HER-2/neu targeted magnetic iron oxide nanoparticles (IONPs) led to about 5-fold contrast enhancements in the tumor for PAT, while near-infrared (NIR) dye labeled nanoparticles emitted strong optical signals for FMT. Both PAT and FMT were demonstrated to be able to detect ovarian tumors located deep in the peritoneal cavity in mice. The targeted nanoprobes allowed mapping tumors in high resolution via PAT, and high sensitivity and specificity via FMT. This study demonstrated the potential of the application of HER-2/neu-targeted PAT/FMT approach for non-invasive or intraoperative imaging of ovarian cancer. FROM THE CLINICAL EDITOR: This paper details the development of a dual-modality photoacoustic and fluorescence molecular tomography approach in combination with a targeted imaging probe for three-dimensional imaging of ovarian tumors in a mouse model, demonstrating the application of the HER-2/neu-targeted approach for non-invasive or intraoperative imaging of ovarian cancer.

Enhanced peritoneal ovarian tumor dissemination by tissue transglutaminase.

Tissue transglutaminase (TG2) is involved in Ca(2+)-dependent aggregation and polymerization of proteins. We previously reported that TG2 mRNA is up-regulated in epithelial ovarian cancer (EOC) cells compared with normal ovarian epithelium. Here, we show overexpression of the TG2 protein in ovarian cancer cells and tumors and its secretion in ascites fluid and define its role in EOC. By stable knockdown and overexpression, we show that TG2 enhances EOC cell adhesion to fibronectin and directional cell migration. This phenotype is preserved in vivo, where the pattern of tumor dissemination in the peritoneal space is dependent on TG2 expression levels. TG2 knockdown diminishes dissemination of tumors on the peritoneal surface and mesentery in an i.p. ovarian xenograft model. This phenotype is associated with deficient beta(1) integrin-fibronectin interaction, leading to weaker anchorage of cancer cells to the peritoneal matrix. Highly expressed in ovarian tumors, TG2 facilitates i.p. tumor dissemination by enhancing cell adhesion to the extracellular matrix and modulating beta(1) integrin subunit expression.

Sequence diverse miRNAs converge to induce mesenchymal-to-epithelial transition in ovarian cancer cells through direct and indirect regulatory controls.

Epithelial-to-mesenchymal transition (EMT) has been shown to be similarly regulated by multiple miRNAs, some displaying little or no sequence identity. While alternate models have been proposed to explain the functional convergence of sequence divergent miRNAs, little experimental evidence exists to elucidate the underlying mechanisms involved. Representative members of the miR-200 family of miRNAs and the sequence divergent miR-205 miRNA were independently over expressed in mesenchymal-like ovarian cancer (OC) cells resulting in mesenchymal-to-epithelial transition (MET). The miR-205 and the miR-200 family of miRNAs were found to coordinately induce MET in mesenchymal-like OC cells by affecting both direct and indirect changes in the expression of genes previously associated with EMT/MET. Only two direct targets of these miRNAs (ZEB 1 and WNT5A) are commonly down regulated in response to over-expression of miR-205 and/or the miR-200 family of miRNAs. Down-regulation of these genes, alone or in combination, only partially recapitulates the changes induced by the miRNAs indicating an additional contribution of indirect changes regulated by the miRNAs. Combined gene expression analyses and phylogenetic comparisons suggest an evolutionarily more recent involvement of miR-205 in the EMT/MET process.

Targeted Drug Delivery and Image-Guided Therapy of Heterogeneous Ovarian Cancer Using HER2-Targeted Theranostic Nanoparticles.

Cancer heterogeneity and drug resistance limit the efficacy of cancer therapy. To address this issue, we have developed an integrated treatment protocol for effective treatment of heterogeneous ovarian cancer. Methods: An amphiphilic polymer coated magnetic iron oxide nanoparticle was conjugated with near infrared dye labeled HER2 affibody and chemotherapy drug cisplatin. The effects of the theranostic nanoparticle on targeted drug delivery, therapeutic efficacy, non-invasive magnetic resonance image (MRI)-guided therapy, and optical imaging detection of therapy resistant tumors were examined in an orthotopic human ovarian cancer xenograft model with highly heterogeneous levels of HER2 expression. Results: We found that systemic delivery of HER2-targeted magnetic iron oxide nanoparticles carrying cisplatin significantly inhibited the growth of primary tumor and peritoneal and lung metastases in the ovarian cancer xenograft model in nude mice. Differential delivery of theranostic nanoparticles into individual tumors with heterogeneous levels of HER2 expression and various responses to therapy were detectable by MRI. We further found a stronger therapeutic response in metastatic tumors compared to primary tumors, likely due to a higher level of HER2 expression and a larger number of proliferating cells in metastatic tumor cells. Relatively long-time retention of iron oxide nanoparticles in tumor tissues allowed interrogating the relationship between nanoparticle drug delivery and the presence of resistant residual tumors by in vivo molecular imaging and histological analysis of the tumor tissues. Following therapy, most of the remaining tumors were small, primary tumors that had low levels of HER2 expression and nanoparticle drug accumulation, thereby explaining their lack of therapeutic response. However, a few residual tumors had HER2-expressing tumor cells and detectable nanoparticle drug delivery but failed to respond, suggesting additional intrinsic resistant mechanisms. Nanoparticle retention in the small residual tumors, nevertheless, produced optical signals for detection by spectroscopic imaging. Conclusion: The inability to completely excise peritoneal metastatic tumors by debulking surgery as well as resistance to chemotherapy are the major clinical challenges for ovarian cancer treatment. This targeted cancer therapy has the potential for the development of effective treatment for metastatic ovarian cancer.

Tissue transglutaminase protects epithelial ovarian cancer cells from cisplatin-induced apoptosis by promoting cell survival signaling.

Tissue transglutaminase (TG2), an enzyme involved in protein cross-linking and overexpressed in ovarian tumors, has antiapoptotic effects in cancer cells and may play a role in response to chemotherapy. In this study, we investigated the role of TG2 in the sensitivity of ovarian cancer cells to cisplatin. By using stable knockdown and overexpression strategies, we demonstrate that the level of expression of TG2 regulates apoptosis induced by cisplatin in SKOV3 and OV-90 ovarian cancer cells. Interestingly, not only TG2 knockdown but also a TG2 enzymatic inhibitor (KCC009) sensitized SKOV3 cells to cisplatin. To understand the mechanism by which TG2 exerts its antiapoptotic role, we examined the effects of protein kinase B (Akt) and nuclear factor-kappa B (NF-kappaB), two survival pathways commonly involved in development of drug resistance. Overexpression of the constitutively active p65 subunit of NF-kappaB, but not constitutively active Akt, rescued cells with diminished TG2 expression from cisplatin-induced apoptosis. This implicates activation of NF-kappaB as the main cisplatin resistance mechanism downstream of TG2. Indeed, NF-kappaB activity is decreased and the level of the inhibitory subunit I kappaB alpha is increased in ovarian cancer cells engineered to express diminished levels of TG2 or treated with the enzymatic inhibitor, KCC009. Our data show that TG2 prevents apoptosis induced by cisplatin by activating the NF-kappaB survival pathway in ovarian cancer cells.

Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells.

PURPOSE: Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. METHODS: Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. RESULTS: Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC=133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA+forskolin: pMLC=88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC=145%; nocodazole: pMLC=145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1+forskolin: pMLC=99%; nocodazole+forskolin: pMLC=107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC=68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. CONCLUSIONS: Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.

Time-course analysis of microRNA-induced mesenchymal-to-epithelial transition underscores the complexity of the underlying molecular processes.

Expression levels of the miR-200 family of miRNAs are significantly reduced during the epithelial-to-mesenchymal transition (EMT) and consequent metastasis of ovarian and other cancers. Consistently, ectopic over-expression of miR-200 family miRNAs in mesenchymal-like cells reverses the process by converting treated cells to an epithelial phenotype, thereby reducing invasiveness and increasing sensitivity to chemotherapeutic drugs. To better understand the dynamics and molecular processes underlying miRNA-induced mesenchymal-to mesenchymal transition (MET), a time-course study was conducted where miRNA-induced morphological and molecular changes associated with MET were monitored over a period of 144 h. Morphological transition from an elongated mesenchymal-like to a cuboidal epithelial-like phenotype is maximized at 48 h with cells returning to the elongated phenotype by 144 h. Changes in the expression of >3000 genes, including many previously associated with epithelial-to-mesenchymal transition (EMT), are most pronounced at 48 h, and approach starting levels of expression by 144 h. The majority of these genes are not direct targets of miR-429. Targeted (siRNA) inhibition of key miR-429 regulated genes previously implicated as drivers of EMT/MET, do not recapitulate miR-429 induced MET indicating that the underlying molecular processes are complex.

Benzalkonium chloride induces dephosphorylation of Myosin light chain in cultured corneal epithelial cells.

PURPOSE: Phosphorylation of myosin light chain (MLC) is essential for the contractility of the actin cytoskeleton, which regulates barrier integrity, adhesion, and migration. This study was conducted to investigate the effect of benzalkonium chloride (BAK), a preservative in topical ophthalmic formulations, on MLC phosphorylation in primary cultures of bovine corneal epithelial cells (BCECs). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by Western blot analysis. Activation of RhoA, which inhibits MLC phosphatase through Rho kinase, was examined by immunoprecipitation. The release of adenosine triphosphate (ATP) was measured by the luciferase-luciferin bioluminescence technique. RESULTS: Positive expression of MLC kinase (MLCK) was found at the mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Exposure to BAK for 10 to 20 minutes at concentrations of 0.0005%, 0.001%, and 0.003% reduced MLC phosphorylation by more than 30%. In addition, BAK led to thinning of the cortical actin and a decrease in cell adhesion. However, RhoA activity was found to increase with BAK treatment. Similar to BAK, ATP-depletion (induced by both antimycin-A and hypoxia) led to MLC dephosphorylation. BAK exposure also showed acute ATP release. CONCLUSIONS: BAK induces acute ATP release and concomitant MLC dephosphorylation in bovine corneal epithelial cells. The dephosphorylation, presumably due to ATP loss, is indicative of a loss of contractility of the actin cytoskeleton that could affect cellular functions contributing to the maintenance of epithelial barrier integrity.

Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells.

PURPOSE: To investigate histamine-induced changes in the phosphorylation of myosin light chain (MLC) and its influence on the barrier integrity of corneal endothelial cells through altered contractility of the actin cytoskeleton. METHODS: Experiments were performed in cultured bovine corneal endothelial cells (BCECs). Phosphorylation of MLC, which increases contractility of the actin cytoskeleton through actomyosin interaction, was assessed by urea-glycerol gel electrophoresis and Western blot analysis. Immunocytochemistry was used to locate phosphorylated MLC in relation to tight junctions. Phosphorylation of the 17-kDa PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17), which inhibits MLC phosphatase, was studied using Western blot analysis. The cortical actin cytoskeleton was visualized by staining with Texas-red phalloidin. Barrier integrity was determined by quantifying horseradish peroxidase (HRP; 44 kDa) flux across cells grown on porous filters. RESULTS: RT-PCR and Western blot analysis confirmed the expression of Galphaq/11-coupled H1 receptors in BCECs. Exposure to histamine (100 microM; 10 minutes) led to phosphorylation of MLC (134% relative to untreated cells) and of CPI-17. Histamine also increased the flux of HRP by sevenfold and disrupted the assembly of the dense cortical actin found in resting cells. PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM; 30 minutes) caused phosphorylation of both MLC and CPI-17. The histamine-induced MLC phosphorylation was reduced by pre-exposure to either ML-7 (50 microM), an MLCK (MLC kinase) inhibitor, or chelerythrine (10 microM), an inhibitor of PKC. Cotreatment with agents that elevate cAMP in BCECs prevented the histamine-induced MLC phosphorylation and the disruption of the actin cytoskeleton, and increased HRP flux. Phosphorylated MLC in response to histamine or PMA was found in a punctate form in close proximity to ZO-1, a marker of the tight junctional complex. CONCLUSIONS: Histamine induces MLC phosphorylation by activating MLCK and partly inhibiting MLC phosphatase. The latter is facilitated by the phosphorylation of CPI-17. Localization of phosphorylated MLC in proximity to ZO-1 suggests increased contractility of the cortical actin at the tight junctional complex. This contractility oppose the tethering forces and lead to a breakdown of the barrier integrity. Last, elevated cAMP prevents histamine-induced loss of the barrier integrity, not only by blocking inactivation of MLC phosphatase but also by inactivating MLCK.

Targeted in vivo delivery of EGFR siRNA inhibits ovarian cancer growth and enhances drug sensitivity.

A functionalized nanohydrogel siRNA delivery system and a mouse model of serous ovarian cancer were used to test predictions from previous cell line studies that knockdown of EGFR (epidermal growth factor receptor) may be of clinical significance in the treatment of epithelial tumors especially with respect to the enhancement of platinum based therapies. Our results support these predictions and suggest that targeted delivery of EGFR siRNA may be an effective strategy for the treatment of ovarian and other epithelial tumors associated with elevated levels of EGFR and especially those demonstrating resistance to platinum-based therapies.

Optical imaging of ovarian cancer using HER-2 affibody conjugated nanoparticles.

Computed Tomography (CT), Ultrasound (US), and Magnetic Resonance Imaging (MRI) have been the mainstay of clinical imaging regimens for the detection of ovarian cancer. However, without tumor specific contrast enhancement, these imaging modalities lack specificity and sensitivity in the detection of small primary and disseminated tumors in the peritoneal cavity. Herein, we illustrate a fairly new near infrared (NIR) optical imaging approach developed in our laboratory for the noninvasive detection of ovarian tumors using a HER-2 targeted nanoparticle-based imaging agent in an orthotopic mouse model of ovarian cancer. We used multimodal imaging approaches to detect the disease accurately and rapidly by utilizing a single imaging agent, NIR dye-labeled HER-2 affibody conjugated iron oxide nanoparticles. This agent targets HER-2 receptors, which are overexpressed in ovarian tumors. This chapter outlines materials and methods for the: (1) production of HER-2 targeted nanoparticles; (2) establishment of an orthotopic human ovarian cancer xenograft model; (3) monitoring of tumor growth by bioluminescence imaging; (4) administration of targeted nanoparticles followed by NIR optical imaging for the detection of orthotopic ovarian cancers with targeted accumulation of the nanoparticle imaging probes.

Theranostic nanoparticles with controlled release of gemcitabine for targeted therapy and MRI of pancreatic cancer.

The tumor stroma in human cancers significantly limits the delivery of therapeutic agents into cancer cells. To develop an effective therapeutic approach overcoming the physical barrier of the stroma, we engineered urokinase plasminogen activator receptor (uPAR)-targeted magnetic iron oxide nanoparticles (IONPs) carrying chemotherapy drug gemcitabine (Gem) for targeted delivery into uPAR-expressing tumor and stromal cells. The uPAR-targeted nanoparticle construct, ATF-IONP-Gem, was prepared by conjugating IONPs with the amino-terminal fragment (ATF) peptide of the receptor-binding domain of uPA, a natural ligand of uPAR, and Gem via a lysosomally cleavable tetrapeptide linker. These theranostic nanoparticles enable intracellular release of Gem following receptor-mediated endocytosis of ATF-IONP-Gem into tumor cells and also provide contrast enhancement in magnetic resonance imaging (MRI) of tumors. Our results demonstrated the pH- and lysosomal enzyme-dependent release of gemcitabine, preventing the drug from enzymatic degradation. Systemic administrations of ATF-IONP-Gem significantly inhibited the growth of orthotopic human pancreatic cancer xenografts in nude mice. With MRI contrast enhancement by IONPs, we detected the presence of IONPs in the residual tumors following the treatment, suggesting the possibility of monitoring drug delivery and assessing drug-resistant tumors by MRI. The theranostic ATF-IONP-Gem nanoparticle has great potential for the development of targeted therapeutic and imaging approaches that are capable of overcoming the tumor stromal barrier, thus enhancing the therapeutic effect of nanoparticle drugs on pancreatic cancers.

Tissue transglutaminase regulates matrix metalloproteinase-2 in ovarian cancer by modulating cAMP-response element-binding protein activity.

Tissue transglutaminase 2 (TG2) is overexpressed in epithelial ovarian cancer (EOC) and promotes intraperitoneal metastasis. How TG2 facilitates the spread of EOC is unknown. Here, we show that TG2 regulates the expression and function of matrix metalloproteinase-2 (MMP-2), a critical mediator of tissue invasiveness. TG2 knockdown down-regulates MMP-2 protein and mRNA expression in SKOV3, IGROV-1, MDA-MB-436, and PC-3 cancer cells. TG2 knockdown or inhibition of TG2 activity using KCC009 decreases MMP-2 gelatinase activity in cancer cells. MMP-2 expression and function are regulated by TG2 at transcriptional level, as demonstrated by quantitative PCR and reporter assays. We used bioinformatics and chromatin immunoprecipitation to identify a CREB binding site in the MMP-2 promoter. Binding of CREB to the MMP-2 promoter was diminished in cells that expressed decreased TG2 levels. TG2 knockdown decreased CREB phosphorylation, and CREB knockdown decreased MMP-2 expression. The effect of TG2 on CREB activity and MMP-2 transcription is mediated by TG2-dependent degradation of protein phosphatase 2 (PP2A-alpha). We show that PP2A-alpha complexes with and is targeted for degradation by TG2. In addition to their related in vitro expression levels, TG2 and MMP-2 expression were significantly correlated in vivo, as shown by concordant immunostaining in peritoneal xenografts and in human ovarian tumors. The capacity of TG2 to regulate MMP-2 expression in vitro and in vivo identifies a mechanism that may facilitate tissue invasion and the spread of EOC. The demonstration that TG2 induced degradation of PP2A-alpha activates CREB, and thereby increases MMP-2 transcription, provides novel mechanistic insight into the pro- metastatic function of TG2.

Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tissue transglutaminase.

Tissue transglutaminase (TG2), an enzyme that catalyzes Ca(2+)-dependent aggregation and polymerization of proteins, is overexpressed in ovarian cancer cells and tumors. We previously reported that TG2 facilitates tumor dissemination using an i.p. xenograft model. Here we show that TG2 modulates epithelial-to-mesenchymal transition (EMT), contributing to increased ovarian cancer cell invasiveness and tumor metastasis. By using stable knockdown and overexpression in epithelial ovarian cancer cells, we show that TG2 induces a mesenchymal phenotype, characterized by cadherin switch and invasive behavior in a Matrigel matrix. This is mediated at the transcriptional level by altering the expression levels and function of several transcriptional repressors, including Zeb1. One mechanism through which TG2 induces Zeb1 is by activating the nuclear factor-kappaB complex. The effects of TG2 on ovarian cancer cell phenotype and invasiveness translate into increased tumor formation and metastasis in vivo, as assessed by an orthotopic ovarian xenograft model. Highly expressed in ovarian tumors, TG2 promotes EMT and enhances ovarian tumor metastasis by activating oncogenic signaling.

Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells.

PURPOSE: To investigate changes in the phosphorylation of myosin light chain (MLC) in response to histamine and its effect on the barrier integrity of corneal epithelial cells. MATERIALS AND METHODS: Experiments were performed in bovine corneal epithelial cells (BCEC). RT-PCR and Western blotting were employed to characterize expression of H1 receptors and MLC kinase (MLCK). Phosphorylation of MLC was assessed by urea-glycerol gel electrophoresis and Western blotting. Barrier integrity was determined as permeability to horseradish peroxidase (HRP; 44 kDa) across monolayers grown on porous filters. RESULTS: Expression of both H1 receptors and MLCK was found in BCEC. Exposure to histamine induced significant MLC phosphorylation concomitant with an increase in HRP permeability. In addition, organization of the cortical actin found in resting cells was disrupted. In contrast to histamine, ATP (a P2Y receptor agonist) induced dephosphorylation of MLC. Pre-exposure to ATP reduced the effect of histamine on HRP permeability and disruption of cortical actin. CONCLUSION: MLC phosphorylation, a biochemical pre-requisite for increased contractility of the actin cytoskeleton, led to histamine-induced breakdown of the barrier integrity in the corneal epithelial cells. This is attributed to weakening of the tethering forces at the tight junctions by the centripetal forces produced by increased actin contractility.

The platelet-derived growth factor receptor alpha is destabilized by geldanamycins in cancer cells.

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.

Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase.

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.