Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.

Curcumin, the active ingredient in Curcuma longa, has been in medicinal use since ancient times. However, the therapeutic targets and signaling cascades modulated by curcumin have been enigmatic despite extensive research. Here we identify dual-specificity tyrosine-regulated kinase 2 (DYRK2), a positive regulator of the 26S proteasome, as a direct target of curcumin. Curcumin occupies the ATP-binding pocket of DYRK2 in the cocrystal structure, and it potently and specifically inhibits DYRK2 over 139 other kinases tested in vitro. As a result, curcumin diminishes DYRK2-mediated 26S proteasome phosphorylation in cells, leading to reduced proteasome activity and impaired cell proliferation. Interestingly, curcumin synergizes with the therapeutic proteasome inhibitor carfilzomib to induce apoptosis in a variety of proteasome-addicted cancer cells, while this drug combination exhibits modest to no cytotoxicity to noncancerous cells. In a breast cancer xenograft model, curcumin treatment significantly reduces tumor burden in immunocompromised mice, showing a similar antitumor effect as CRISPR/Cas9-mediated DYRK2 depletion. These results reveal an unexpected role of curcumin in DYRK2-proteasome inhibition and provide a proof-of-concept that pharmacological manipulation of proteasome regulators may offer new opportunities for anticancer treatment.

Mitochondrial-Targeted Decyl-Triphenylphosphonium Enhances 2-Deoxy-D-Glucose Mediated Oxidative Stress and Clonogenic Killing of Multiple Myeloma Cells.

Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM) but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266), using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG) induced ER stress as detected by qPCR (BiP, ATF4) and immunoblotting (BiP, CHOP) and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM cells via 2-DG and 10-TPP combination therapy.

Copper-zinc superoxide dismutase-mediated redox regulation of bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper-zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity.

EGFR inhibition induces proinflammatory cytokines via NOX4 in HNSCC.

UNLABELLED: Chronic inflammation plays a fundamental role in tumor promotion, migration, and invasion. With the use of microarray profiling, a profound increase was observed for those transcripts involved in proinflammatory signaling in epidermal growth factor receptor (EGFR) inhibitor-treated head and neck squamous cell carcinoma (HNSCC) cells as compared with their respective controls. As such, it was hypothesized that EGFR inhibitor efficacy is offset by the proinflammatory response that these therapeutics conjure in HNSCC. Systematic evaluation of the clinical EGFR inhibitors-erlotinib, cetuximab, lapatinib, and panitumumab-revealed increased secretion of proinflammatory cytokines such as interleukins (IL-2, IL-4, IL-6, IL-8), granulocyte-macrophage colony-stimulating factor, TNF-α, and IFN-γ. Mechanistic focus on IL-6 revealed that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression. Importantly, exogenous IL-6 protected HNSCC cells from erlotinib-induced cytotoxicity, whereas tocilizumab, an IL-6 receptor antagonist, sensitized cells to erlotinib in vitro and in vivo. Inhibitors of NF-κB, p38, and JNK suppressed erlotinib-induced IL-6 expression, suggesting critical roles for NF-κB and MAPK in IL-6 regulation. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced proinflammatory cytokine expression. Taken together, these results demonstrate that clinical EGFR inhibitors induce the expression of proinflammatory cytokines via NOX4. IMPLICATIONS: The antitumor activity of EGFR inhibitors is reduced by activation of NOX4-mediated proinflammatory pathways in HNSCC.

Scavenger receptor class A member 3 (SCARA3) in disease progression and therapy resistance in multiple myeloma.

This study evaluates the role of scavenger receptor class A member 3 (SCARA3) in multiple myeloma (MM). SCARA3 expression was induced upon treatment with oxidative stressors (ionizing radiation and chemotherapeutic drugs). An epigenetic inactivation of SCARA3 was noted in MM.1S myeloma cells. Myeloma cell killing by dexamethasone and bortezomib was inhibited by up-regulation of SCARA3 while SCARA3 knockdown sensitized myeloma cells to the drugs. Clinical samples showed an inverse correlation between SCARA3 gene expression, myeloma progression, and favorable clinical prognosis. In MM, SCARA3 protects against oxidative stress-induced cell killing and can serve as predictor of MM progression and therapeutic response.

Combination chemotherapy increases cytotoxicity of multiple myeloma cells by modification of nuclear factor (NF)-κB activity.

The nuclear factor (NF)-κB signaling pathway is critical in myeloma cell proliferation, inhibition of apoptosis, and emergence of therapy resistance. The chemotherapeutic drugs, dexamethasone (Dex) and bortezomib (BTZ), are widely used in clinical protocols for multiple myeloma (MM) and inhibit the NF-κB signaling pathway by distinct mechanisms. This study evaluates the efficacy of combination therapy with Dex and BTZ and investigates the mechanistic underpinning of endogenous and therapy-induced NF-κB activation in MM. Human myeloma cells and bone marrow stromal cells (BMSCs) were used in monocultures and cocultures to determine the cytotoxic effects of Dex and/or BTZ. Our results show that combined treatment of Dex with BTZ enhanced direct apoptosis of drug-sensitive and drug-resistant myeloma cells. In the presence of BMSCs, Dex plus BTZ combination inhibited ionizing radiation-induced interleukin 6 secretion from BMSCs and induced myeloma cytotoxicity. Mechanistically, Dex treatment increased IκBα protein and mRNA expression and compensated for BTZ-induced IκBα degradation. Dex plus BTZ combination inhibited basal and therapy-induced NF-κB activity with cytotoxicity in myeloma cells resistant to BTZ. Furthermore, combination therapy downregulated the NF-κB-targeted gene expression of interleukin 6 and manganese superoxide dismutase, which can induce chemo- and radio-resistance in MM. This study provides a mechanistic rationale for combining the NF-κB-targeting drugs Dex and BTZ in myeloma therapy and supports potential combinations of these drugs with radiotherapy and additional chemotherapeutic drugs for clinical benefit in MM.

Secretion of soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt1) requires Arf1, Arf6, and Rab11 GTPases.

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.

Interleukin-6 counteracts therapy-induced cellular oxidative stress in multiple myeloma by up-regulating manganese superoxide dismutase.

IL (interleukin)-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance, but the resistance mechanisms remain unclear. The present study determines the role of IL-6 in re-establishing intracellular redox homoeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress, including IR (ionizing radiation) and Dex (dexamethasone). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase 3 activation, PARP [poly(ADP-ribose) polymerase] cleavage and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early intracellular pro-oxidant levels that were causally related to activation of NF-κB (nuclear factor κB) as determined by the ability of N-acetylcysteine to suppress both pro-oxidant levels and NF-κB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF (tumour necrosis factor)-α, IL-6 induced an early perturbation in reduced glutathione level and increased NF-κB-dependent MnSOD (manganese superoxide dismutase) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. The present study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. The results of the present study indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.

Manipulation of cellular redox parameters for improving therapeutic responses in B-cell lymphoma and multiple myeloma.

Developing novel combined-modality therapeutic approaches based on understanding of the involvement of redox biology in apoptosis of malignant cells is a promising approach for improving clinical responses in B-cell lymphoma and multiple myeloma. Therapeutic modalities that generate reactive oxygen species (i.e., radiation, photodynamic therapy, and specific chemotherapeutic drugs) have been shown to be selectively cytotoxic to malignant B-cells. In this review, we will discuss agents that induce apoptosis in B-cell tumors by oxidative stress. Subsequently, a novel biochemical rationale (based on fundamental differences in cancer vs. normal cell oxidative metabolism) for combining oxidative stressors with radiotherapy and chemotherapy, that may lead to designing of more effective treatment strategies for B-cell malignancies, will be discussed. Besides providing potential curative benefit, such novel therapies could also selectively target and inhibit the emergence of drug-resistance in tumor cells, which is a major determinant of treatment failure in many B-cell malignancies.

Endothelial cell migration on fibronectin is regulated by syntaxin 6-mediated alpha5beta1 integrin recycling.

The α5ß1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5ß1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5ß1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of ß1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5ß1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.

Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6.

Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.

Dexamethasone-induced oxidative stress enhances myeloma cell radiosensitization while sparing normal bone marrow hematopoiesis.

Dexamethasone (Dex) and radiation therapy are established modalities in multiple myeloma. In this study, we propose a novel combination of Dex plus radiation that shows superior clonogenic cell killing and apoptosis of myeloma cells and selectively eliminates myeloma cells when cocultured with bone marrow stromal cells (BMSCs). Dex was found to inhibit the release of interleukin-6 from irradiated BMSCs, which is an established myeloma cell proproliferative cytokine. In 5TGM1 model, the combination of Dex with skeletal targeted radiotherapy (153-Sm-EDTMP) prolonged median survival time and inhibited radiation-induced myelosuppression. A two-cycle treatment of Dex plus 153-Sm-EDTMP was well tolerated and further improved median survival time. Mechanistically, Dex increased superoxide and hydrogen peroxide production and augmented radiation-induced oxidative stress and cell death of myeloma cells. In contrast, Dex inhibited radiation-induced increase in pro-oxidant levels and enhanced the clonogenic survival in normal hematopoietic stem and progenitor cells. Treatment with either N-acetylcysteine or the combination of polyethylene glycol (PEG)-conjugated copper, zinc-superoxide dismutase, and PEG-catalase significantly protected myeloma cells from Dex-induced clonogenic death. Overall, these results demonstrate that Dex in combination with radiotherapy enhances the killing of myeloma cells while protecting normal bone marrow hematopoiesis through a mechanism that involves selective increases in oxidative stress.

Pleiotropic effects of p300-mediated acetylation on p68 and p72 RNA helicase.

Here, we demonstrate that p68 (DDX5) and p72 (DDX17), two homologous RNA helicases and transcriptional cofactors, are substrates for the acetyltransferase p300 in vitro and in vivo. Mutation of acetylation sites affected the binding of p68/p72 to histone deacetylases, but not to p300 or estrogen receptor. Acetylation additionally increased the stability of p68 and p72 RNA helicase and stimulated their ability to coactivate the estrogen receptor, thereby potentially contributing to its aberrant activation in breast tumors. Also, acetylation of p72, but not of p68 RNA helicase, enhanced p53-dependent activation of the MDM2 promoter, pointing at another mechanism of how p72 acetylation may facilitate carcinogenesis by boosting the negative p53-MDM2 feedback loop. Furthermore, blocking p72 acetylation caused cell cycle arrest and apoptosis, revealing an essential role for p72 acetylation. In conclusion, our report has identified for the first time that acetylation modulates RNA helicases and provides multiple mechanisms how acetylation of p68 and p72 may affect normal and tumor cells.

Radioiodide imaging and radiovirotherapy of multiple myeloma using VSV(Delta51)-NIS, an attenuated vesicular stomatitis virus encoding the sodium iodide symporter gene.

Multiple myeloma is a radiosensitive malignancy that is currently incurable. Here, we generated a novel recombinant vesicular stomatitis virus [VSV(Delta51)-NIS] that has a deletion of methionine 51 in the matrix protein and expresses the human sodium iodide symporter (NIS) gene. VSV(Delta51)-NIS showed specific oncolytic activity against myeloma cell lines and primary myeloma cells and was able to replicate to high titers in myeloma cells in vitro. Iodide uptake assays showed accumulation of radioactive iodide in VSV(Delta51)-NIS-infected myeloma cells that was specific to the function of the NIS transgene. In bg/nd/xid mice with established subcutaneous myeloma tumors, administration of VSV(Delta51)-NIS resulted in high intratumoral virus replication and tumor regression. VSV-associated neurotoxicity was not observed. Intratumoral spread of the infection was monitored noninvasively by serial gamma camera imaging of (123)I-iodide biodistribution. Dosimetry calculations based on these images pointed to the feasibility of combination radiovirotherapy with VSV(Delta51)-NIS plus (131)I. Immunocompetent mice with syngeneic 5TGM1 myeloma tumors (either subcutaneous or orthotopic) showed significant enhancements of tumor regression and survival when VSV(Delta51)-NIS was combined with (131)I. These results show that VSV(Delta51)-NIS is a safe oncolytic agent with significant therapeutic potential in multiple myeloma.

Synergistic activity of the proteasome inhibitor PS-341 with non-myeloablative 153-Sm-EDTMP skeletally targeted radiotherapy in an orthotopic model of multiple myeloma.

Multiple myeloma is a highly radiosensitive skeletal malignancy, but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, 2 doses of PS-341 (0.5 mg/kg), or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22, and 28 days, respectively. In contrast, mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg), 1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P < .001). In addition to prolonged survival, this treatment combination yielded reduced clonogenicity of bone marrow-resident 5TGM1 cells, reduced serum myeloma-associated paraprotein levels, and better preservation of bone mineral density. Myelosuppression, determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors, did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent, selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma.

PS-341-mediated selective targeting of multiple myeloma cells by synergistic increase in ionizing radiation-induced apoptosis.

OBJECTIVE: Multiple myeloma remains incurable with current therapy. The proteosome inhibitor, PS-341, has shown objective clinical responses in relapsed refractory myeloma patients. We investigated the potential of enhancing the radiosensitivity of myeloma cells by combining with PS-341; the underlying mechanisms were delineated. MATERIALS AND METHODS: Clonogenic assays were used to evaluate cell survival after exposure to PS-341, ionizing radiation (IR), or PS-341 followed by IR. Apoptosis was studied by annexin V-propidium iodide staining and caspase activation. Cell-cycle phase distribution of cells was determined. Nuclear factor-kappaB (NF-kappaB) activity was monitored by enzyme-linked immunosorbent assay and Western blotting. The expression of death receptor Fas/APO-1/CD95 was analyzed by flow cytometry. The consequential caspase-8 activation was detected by Western blotting. RESULTS: In clonogenic assays, sequential exposure to nontoxic doses of PS-341 (10 nM) and IR (6 Gy) resulted in synergistic inhibition of proliferation of myeloma cells by modulating the apoptotic sensitivity of these cells. Biochemically, sublethal dose of IR led to potent induction of NF-kappaB activity, and this response was significantly inhibited by pretreatment with PS-341, or by the NF-kappaB inhibitory peptide SN-50. Enhanced Fas expression was seen in myeloma cells exposed sequentially to PS-341 and IR. Finally, PS-341 sensitized primary myeloma (CD138+ve) cells to IR but had little effect on CD138-ve bone marrow cells from myeloma patients. CONCLUSION: These data indicate that PS-341 can sensitize myeloma cells to IR by both intrinsic and extrinsic apoptotic pathways. The study indicates improved therapeutic benefits in treatment of multiple myeloma by combining PS-341 with conventional radiotherapy.

Engineering and characterization of a divalent single-chain Fv angiotensin II fusion construct of the monoclonal antibody CC49.

For the therapy of solid tumors, co-administration of angiotensin II (AngII) results in an increased uptake of drugs into the tumor interstitium. We have engineered a dimeric sc(Fv)(2)-AngII fusion construct that combines the superior kinetics of covalent dimeric scFvs [sc(Fv)(2)], recognizing the pancarcinoma tumor-associated antigen 72 (TAG-72), with the advantageous intrinsic activity of AngII. The binding characteristics of the fusion construct were unaltered by the addition of the AngII sequence [affinity constant K(A) 1.18 x 10(7) and 8.42 x 10(6) M(-1) for sc(Fv)(2) and sc(Fv)(2)-AngII, respectively]. The binding of the fusion construct to the angiotensin receptor (AT(1)) was similar to AngII, and the arterial contraction was 16 +/- 1% of the response observed with norepinephrine. In animal studies, the radiolabeled sc(Fv)(2)-AngII construct exhibited similar uptake and a more homogeneous distribution within the tumor as compared to sc(Fv)(2).

The in vivo characteristics of genetically engineered divalent and tetravalent single-chain antibody constructs.

Engineered multivalent single-chain Fv (scFv) constructs have been demonstrated to exhibit rapid blood clearance and better tumor penetration. To understand the short plasma half-life of multivalent single-chain antibody fragments, the pharmacokinetic properties of covalent dimeric scFv [sc(Fv)2], noncovalent tetrameric scFv {[sc(Fv)2]2} and IgG of MAb CC49 were examined. The scFvs displayed an ability to form higher molecular aggregates in vivo. A specific proteolytic cleavage of the linker sequence of the covalent dimeric or a deterioration of the noncovalent association of the dimeric scFv into tetravalent scFv constructs was not observed. In conclusion, sc(Fv)2 and [sc(Fv)2]2 are stable in vivo and have significant potential for diagnostic and therapeutic applications.

Concerted activation of ETS protein ER81 by p160 coactivators, the acetyltransferase p300 and the receptor tyrosine kinase HER2/Neu.

Activator of thyroid and retinoic acid receptor (ACTR) is overexpressed in approximately 60% of primary human breast tumors and belongs to the p160 steroid receptor coactivator family. In this study, we identified a novel interaction partner of ACTR, the ETS transcription factor ER81 that is also heavily implicated in mammary tumor formation. ACTR and related p160 family members (steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein-1 (GRIP-1)) augment ER81-mediated transcription. Although ACTR and GRIP-1 can acetylate ER81, this posttranslational modification of ER81 is not required for its stimulation by ACTR or GRIP-1. In addition, ACTR collaborates with the p300 coactivator, a joint interaction partner of ACTR and ER81, to stimulate ER81 function and the ability of p300 to acetylate ER81 is indispensable for this collaboration. Furthermore, the receptor tyrosine kinase HER2/Neu, an oncoprotein particularly found overexpressed in breast tumors, cooperates with both ACTR and p300 to stimulate ER81-mediated transcription. Thus, oncogenic HER2/Neu and ACTR may synergize to orchestrate mammary tumorigenesis through the dysregulation of the transcription factor ER81 and its target genes.