12 research outputs found
Regulation of α5ÎČ1 integrin conformation and function by urokinase receptor binding
Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with ÎČ1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the ÎČ-propeller of α3ÎČ1 empowers vitronectin adhesion by this integrin. How uPAR modifies other ÎČ1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5ÎČ1 and rather than blocking, renders fibronectin (Fn) binding by α5ÎČ1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5ÎČ1âuPAR to Fn type III repeats 12â15 in addition to type III repeats 9â11 bound by α5ÎČ1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5ÎČ1 conformation. A ÎČ1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a ÎČ1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5ÎČ1. Direct binding and regulation of α5ÎČ1 by uPAR implies a modified âbentâ integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands
Secondary somatic mutations restoring RAD51C and RAD51D associated with acquired resistance to the PARP inhibitor rucaparib in high-grade ovarian carcinoma
High-grade epithelial ovarian carcinomas (OC) containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pre-treatment and post-progression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed OC. In six of 12 pre-treatment biopsies, a truncation mutation in BRCA1, RAD51C or RAD51D was identified. In five of six paired post-progression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft (PDX), as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations
Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.
Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance
Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells
7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced
in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods
based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with
A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation),
mass spectrometry (MALDIâTOF, ESâTOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The
light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other
recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine
297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding
to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a
mixture of three families of antibodies corresponding (i) to the expected structure (17%; 149 297 Da; 1330 amino acids), (ii) a variant with a
translated intron in one heavy chains (33%; 152 878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%;
154 459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal
antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also
identified by peptide mapping, mass spectrometry and microsequencing
Chikungunya Virus Infection during Pregnancy, RĂ©union, France, 2006
Except for prenatal hospital admissions for those infected, this virus had no effect on outcomes
Murine Cathepsin F Deficiency Causes Neuronal Lipofuscinosis and Late-Onset Neurological Disease
Cathepsin F (cat F) is a widely expressed lysosomal cysteine protease whose in vivo role is unknown. To address this issue, mice deficient in cat F were generated via homologous recombination. Although cat F(â/â) mice appeared healthy and reproduced normally, they developed progressive hind leg weakness and decline in motor coordination at 12 to 16 months of age, followed by significant weight loss and death within 6 months. cat F was found to be expressed throughout the central nervous system (CNS). cat F(â/â) neurons accumulated eosinophilic granules that had features typical of lysosomal lipofuscin by electron microscopy. Large amounts of autofluorescent lipofuscin, characteristic of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL), accumulated throughout the CNS but not in visceral organs, beginning as early as 6 weeks of age. Pronounced gliosis, an indicator of neuronal stress and neurodegeneration, was also apparent in older cat F(â/â) mice. cat F is the only cysteine cathepsin whose inactivation alone causes a lysosomal storage defect and progressive neurological features in mice. The late onset suggests that this gene may be a candidate for adult-onset NCL
Multimodal Microvascular Imaging Reveals that Selective Inhibition of Class I PI3K Is Sufficient to Induce an Antivascular Response
The phosphatidylinositol 3-kinase (PI3K) pathway is a central mediator of vascular endothelial growth factor (VEGF)-driven angiogenesis. The discovery of small molecule inhibitors that selectively target PI3K or PI3K and mammalian target of rapamycin (mTOR) provides an opportunity to pharmacologically determine the contribution of these key signaling nodes in VEGF-A-driven tumor angiogenesis in vivo. This study used an array of microvascular imaging techniques to monitor the antivascular effects of selective class I PI3K, mTOR, or dual PI3K/ mTOR inhibitors in colorectal and prostate cancer xenograft models. Micro-computed tomography (micro-CT) angiography, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), vessel size index (VSI) MRI, and DCE ultrasound (DCE-U/S) were employed to quantitatively evaluate the vascular (structural and physiological) response to these inhibitors. GDC-0980, a dual PI3K/mTOR inhibitor, was found to reduce micro-CT angiography vascular density, while VSI MRI demonstrated a significant reduction in vessel density and an increase in mean vessel size, consistent with a loss of small functional vessels and a substantial antivascular response. DCE-MRI showed that GDC-0980 produces a strong functional response by decreasing the vascular permeability/perfusion-related parameter, Ktrans. Interestingly, comparable antivascular effects were observed for both GDC-980 and GNE-490 (a selective class I PI3K inhibitor). In addition, mTOR-selective inhibitors did not affect vascular density, suggesting that PI3K inhibition is sufficient to generate structural changes, characteristic of a robust antivascular response. This study supports the use of noninvasive microvascular imaging techniques (DCE-MRI, VSI MRI, DCE-U/S) as pharmacodynamic assays to quantitatively measure the activity of PI3K and dual PI3K/mTOR inhibitors in vivo