Total Cell Lysates Research Articles

Abstract 805: Intra-tumoral pharmacodynamics of selumetinib in serial biopsies from patients with neurofibroma

Abstract Background: The oral MEK 1/2 inhibitor selumetinib (50-mg po, BID) has shown remarkable response rates in an ongoing phase-II trial (NCT02407405) in adult patients (pts) with neurofibromatosis type-1 (NF1) (cutaneous neurofibroma (cNF) or inoperable plexiform neurofibromas (PN). In this study, we are evaluating the pharmacodynamics (PD) of selumetinib-induced modulation of key phosphorylation sites on MEK1, MEK2, ERK1, and ERK2 using a new, validated, fit-for-purpose multiplex immunoassay of tumor biopsy extracts that distinguishes specific isoforms. Methods: Core needle tumor biopsies scheduled at baseline and pre-cycle 3 of selumetinib treatment were obtained from patients with a confirmed diagnosis of cNF or PN, snap-frozen in liquid nitrogen within 2 min at point of collection, and stored at -80°C. Additional dermal biopsies were collected (6 of 17 pts) to assess clinical suitability of the proposed PD analysis. Total cell lysates of biopsies were prepared in accordance with the NCI SOP that has been validated to preserve phospho-biomarker integrity (PMID 27001313). Samples were analyzed in a single batch on a Luminex platform, and the ratio of phosphorylated to total protein calculated individually for MEK1, MEK 2, ERK1, and ERK2 and compared by pair-wise analysis. Results: The current PD evaluation resulted from analysis of 24 paired biopsies (17 pts; 6 cNF, 18 PN) representing a range in time interval between previous dose and biopsy procedure (43 min - to 28 hrs; median 7.6, 95% CI 3.25 - 13.98 hrs). Selumetinib treatment decreased the median pERK1 ratio by 59% (95% CI: -30 to -77%; p<0.05) and the pERK2 ratio by 58% (95% CI: -27 to -67%; p<0.05). Biomarker variability was higher in cNF biopsies than PN core biopsies. The pERK1 and pERK2 ratios correlated moderately with elapsed time between previous dose and biopsy procedure (p < 0.05) but did not correlate with baseline MEK1 or MEK2 ratios. Selumetinib treatment decreased pERK1 and pERK2 ratios in 15/17 pts, and achieved >50% suppression in 12/17 pts. The last dose maintained pERK1/2 suppression (>50% decrease in their ratio values) for >8 hrs in 5 pts and >20 hrs in 2 pts. Conclusions: Our PD study provides the first evidence of engagement and control of selumetinib's molecular target in NF1. Importantly, molecular target response was demonstrable in 14 of 15 patients who responded clinically to this treatment regimen, a finding enabled by a validated high-performance assay for phosphoproteins that are key PD biomarkers of selumetinib mechanism of action and that may be useful in assessing molecular drug action of other members of this drug class. Funded by NCI Contract No HHSN261200800001E. Citation Format: William Herrick, Geraldine O'Sullivan Coyne, Andrea Gross, Eva Dombi, Jeevan Govindharajulu, Casey Kilpatrick, Jennifer Desanto, Amanda Marie Carbonell, Joanne Derdak, Dominique Corinne Pichard, Cecilia Maura Tibery, Bradford J. Wood, Lawrence Doyle, James H. Doroshow, Alice Chen, Ralph E. Parchment, Brigitte C. Widemann, Apurva K. Srivastava. Intra-tumoral pharmacodynamics of selumetinib in serial biopsies from patients with neurofibroma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 805.

Human Keratinocytes Synthesize Ultra Long‐Chain Sphingoid Bases (Up To C28) That Are Abundant in Skin Stratum Corneum

Skin sphingolipids are unique and fulfill the important task of forming a hydrophobic barrier to limit water loss from the body but also to protect from allergen, bacterial and viral penetration through the skin. Skin sphingolipids are highly diverse in their combination of fatty acids and sphingoid bases, the presence of highly hydrophobic ω‐esterified ceramides (EOS‐CER), and the abundance of ultra long‐chain fatty acids (ULCFA, up to C36) within ceramide molecules. The chain length of sphingoid bases in ceramides is commonly attributed to C18 molecules but there are only a few reports demonstrating the presence of C20‐ and C22‐sphingoid bases in skin ceramides. As free sphingoid bases and ceramides are metabolically connected, it is assumed that similar to other tissues, C18‐sphingoid bases represent the majority of sphingoid bases within the total pool of free and ceramide‐linked molecules in the skin.Our sphingolipidomic work during the study of the mechanisms leading to the impairment of skin barrier function has revealed that ceramides with C18‐, C20‐, and C22‐ sphingoid bases are almost equally represented in human skin stratum corneum. Surprisingly, we found that free sphingoid bases in stratum corneum are highly abundant and composed primarily of ultra long‐chain (C24‐ and C26‐) dihydrosphingosines. This finding prompted us to investigate in details the ability of skin keratinocytes to synthesize ultra long‐chain length sphingoid bases. Using undifferentiated and Ca2+‐differentiated primary human keratinocytes, [U‐13C,15N]‐serine, and stable isotope pulse‐labeling approach in vitro, we have confirmed that all ceramides and free sphingoid base molecules do incorporate the labeled serine into their structure. Next, we explored the ability of serine palmitoyltransferase (SPT), the enzyme that initiates sphingolipid biosynthesis, to synthesize ultra long‐chain sphingoid bases in vitro. By comparing the activity of SPT in total cell lysates prepared from undifferentiated and Ca2+‐differentiated human keratinocytes, 16:0–24:0‐Coenzyme A molecules and [U‐13C,15N]‐serine, we confirmed the ability of SPT expressed in human keratinocytes to produce, albeit at low capacity, C24‐ and C26‐3‐keto‐dihydrosphingosines. As keratinocytes and skin stratum corneum do not have measurable amounts of ceramides with C24‐C26‐dihydrosphingosines or C24‐C26‐sphingosines, we conclude that keratinocyte ceramide synthases are unable to utilize dihydrosphingosines with carbon chain length more than C22, thus providing a foundation for the accumulation of C24‐C26‐dihydrosphingosine in human stratum corneum. The question of the possible special biological function of long‐chain dihydrosphingosines in human skin is currently under investigation.

Novel indole derivatives targeting HuR-mRNA complex to counteract high glucose damage in retinal endothelial cells

The ELAVL1 (or human antigen R - HuR) RNA binding protein stabilizes the mRNA, with an AU-rich element, of several genes such as growth factors (i.e. VEGF) and inflammatory cytokines (i.e. TNFα). We hereby carried out a virtual screening campaign in order to identify and test novel HuR-mRNA disruptors. Best-scored compounds were tested in an in-vitro model of diabetic retinopathy, namely human retinal endothelial cells (HRECs) challenged with high-glucose levels (25 mM). HuR, VEGF and TNFα protein contents were evaluated by western-blot analysis in total cell lysates. VEGF and TNFα released from HRECs were measured in cell medium by ELISA. We found that two derivatives bearing indole moiety, VP12/14 and VP12/110, modulated HuR expression and decreased VEGF and TNF-α release by HREC exposed to high glucose (HG) levels. VP12/14 and VP12/110 inhibited VEGF and TNF-α release in HRECs challenged with high glucose levels, similarly to dihydrotanshinone (DHTS), a small molecule known to interfere with HuR- TNFα mRNA binding. The present findings demonstrated that VP12/14 and VP12/110 are innovative molecules with anti-inflammatory and anti-angiogenic properties, suggesting their potential use as novel candidates for treatment of diabetic retinopathy.

MS1-Level Proteome Quantification Platform Allowing Maximally Increased Multiplexity for SILAC and In Vitro Chemical Labeling.

Quantitative proteomic platforms based on precursor intensity in mass spectrometry (MS1-level) uniquely support in vivo metabolic labeling with superior quantification accuracy but suffer from limited multiplexity (≤3-plex) and frequent missing quantities. Here we present a new MS1-level quantification platform that allows maximal multiplexing with high quantification accuracy and precision for the given labeling scheme. The platform currently comprises 6-plex in vivo SILAC or in vitro diethylation labeling with a dedicated algorithm and is also expandable to higher multiplexity (e.g., nine-plex for SILAC). For complex samples with broad dynamic ranges such as total cell lysates, our platform performs highly accurately and free of missing quantities. Furthermore, we successfully applied our method to measure protein synthesis rate under heat shock response in human cells by 6-plex pulsed SILAC experiments, demonstrating the unique biological merits of our in vivo platform to disclose translational regulations for cellular response to stress.

Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and β isoforms to study their interaction with amyloid β peptide (Aβ42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/β and immobilized Aβ42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Cytolytic Activity of Effector T-lymphocytes Against Hepatocellular Carcinoma is Improved by Dendritic Cells Pulsed with Pooled Tumor Antigens

Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC.

Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1207 unique proteoforms derived from 484 genes.

659 Towards functional analysis of prolylcarboxypeptidase in human skin - a regulator of alpha-MSH activity?

Prolylcarboxypeptidase (PRCP) is s serine protease that recently emerged as a regulator of the central melanocortin system, i. e. control of food intake and energy metabolism, by degrading α-MSH (1-13) into biologically inactive α-MSH (1-12). Therefore, we wondered if PRCP may also play a role in regulation of the peripheral melanocortin system. As a first step towards answering this question we examined the expression of PRCP in various cutaneous cell types at RNA and protein level in vitro. PRCP transcripts were detected in normal human melanocytes, normal human keratinocytes and human dermal fibroblasts (HDFs) from different donors as shown by endpoint reverse transcriptase- polymerase chain reaction (RT-PCR). However, Western immunoblotting revealed expression of PRCP protein only in HDFs but not in the other cell types. Here, PRCP could be visualized as a granular cytoplasmic staining as shown by immunofluorescence analysis. As detected by liquid chromatography coupled with mass spectrometry, HDFs treated with alpha-MSH formed alpha-MSH (1-12) in conditioned media suggesting expression and secretion of functionally active PRCP. Interestingly, quantitative real-time RT-PCR analysis further revealed a time-dependent upregulatory effect of both tumor necrosis factor and ultraviolet A irradiation but not interleukin-1beta on PRCP mRNA in HDFs. However, Western immunoblotting of total cell lysates and detection of PRCP in cell culture supernatants employing ELISA did not reveal upregulation of this enzyme in HDFs exposed to these stressors. Immunohistochemical studies of normal and diseased human skin further revealed that PRCP is mainly expressed in neutrophilic granulocytes, endothelial cells and activated dermal fibroblasts, i. e. during wound healing and in hypertrophic scars but not within the epidermis. Further studies are underway to further define the biological role of PRCP in such conditions.

MdCDPM: A Mass Defect-Based Chemical-Directed Proteomics Method for Targeted Analysis of Intact Sialylglycopeptides.

Protein sialylation is ubiquitous and essential in a wide range of biological processes. Herein, a mass defect-based chemical-directed proteomics method (MdCDPM) was presented for targeted analysis of intact sialylglycopeptides (SGPs). The process starts by specific oxidation of dihydroxy in sialic acid to aldehyde, which was then chemically labeled by two arginine isotopologues (Arg-15N4 and Arg-D4, differs by 36 mDa). The equally mixed precursor partners, spacing tens of mDa apart, enable the direct recognition of SGPs in MS1 level and benefit the subsequent targeted MS2 characterization. The mass envelope of two labeled forms falling into a narrow m/z window strengthens recognition uniqueness greatly, and the proposed 1:1 intensity ratio of doublets will not be readily distorted. More important, such subtle mass differences permit multiple sialic acids labeling without additional complexity of precursor patterns. Also, the partner m/z shifts detail the number of sialic acids contained in the precursor species. By applying MdCDPM, femtomole quantities of SGPs could be detected from total cell lysates, even at a signal-to-noise ratio of as low as 3:1. In addition, assays were performed to estimate the false positive rate and demonstrated high confidence of MdCDPM. Furthermore, it was designed and successfully exploited to analyze SGPs in human serum, which highlighted the feasibility of this strategy for biological applications.

The Effects of Insulin and Retinoic Acid on the Content of Glucose Transporter 4 in Different Preparation Lysates of L6 Muscle Cells (P15-013-19)

ObjectivesGlucose enters the skeletal muscle cells through glucose transporters (GLUTs), a process that is stimulated by insulin through the movement of GLUT4 to the cell membrane. Here, we studied the effects of retinoic acid (RA) and insulin on GLUT4 expression in L6 muscle cells. MethodsRat L6 muscle cells were induced to differentiation after confluency. Cells were incubated in medium containing 2% HS containing in the absence or presence of 1 μM RA without or with 10 nM insulin for 4 or 6 days with replacement of fresh media every two days. Cells were lyzed and prepared in the following ways. 1) Cells were lyzed in lysis buffer (50 mM HEPES pH 7.5, 10 mM EDTA, 10% Glycerol, 2% NP-40 and 2% Triton X-100) on ice for 20 minutes, vortex cells every 10 minutes to help lyse completely. After that, the lysate was centrifuged at 12,000 x g for 15 minutes at 4°C for the collection of both the supernatant and pellet. 2) Cells were directly lyzed in 1 x SDS loading buffer (designated as total cell lysate here). The GLUT4 protein levels in those samples were determined via Western Blot using anti-GLUT4 antibody (#07-1404, C-terminus) from EMD Millipore Corp. ResultsOn day 4 or 6, the supernatants of cells treated with RA + insulin had a significantly less GLUT4 expression that that of the control group. Additional, on day 4, the supernatant of cells treated with insulin alone also had less GLUT4 protein than that of the control group. The pellets of cells on day 4 treated with RA had a significantly less GLUT4 protein level than that of the control group. On day 4 or 6, the GLUT4 protein levels in total cell lysates were not significantly different among the four treatment groups. ConclusionsThe current lysis buffer allowed us to observe that the treatments of RA and/or insulin can significantly affect GLUT4 protein levels in the supernatants. Given the fact that a significant amount of GLUT4 protein remained in the pellets, the lysis buffer used here could not completely solubilize differentiated L6 cells, suggesting that cautions lysis buffers and methods were chosen carefully to prepare L6 cells for the analysis of GLUT4 protein. Additionally, it also indicates that the differentially solubilized fractions probably provide us tools to study the effects of RA and insulin on the subcellular movement of GLUT4 in L6 muscle cells. Funding SourcesThe University of Tennessee, Knoxville.

A Role for Low Density Lipoprotein Receptor Related Protein 1 (LRP1) and Hepatocyte Nuclear Factor 4α (HNF4α) in Regulating Lipid Metabolism

IntroductionLiver is essential for maintaining healthy lipid, carbohydrate, and protein levels. The low density lipoprotein receptor related protein 1 (LRP1) is a multifunctional cell surface receptor that is expressed ubiquitously throughout the body on a variety of cell types. In liver, LRP1 is present on stellate cells, Kupffer cells, biliary cells, and hepatocytes (HEPs), where it is known to be one of the most abundant hepatocellular proteins. Among its many roles, LRP1 is believed to participate in lipoprotein metabolism. Recently, we found that aged mice with a conditional knockout (cKO) for hepatocellular LRP1 (LRP1−) are positive for LRP1 in HEPs that surround the periportal region. Further studies determined that a minor population of LRP1+ cells from younger cKO mice likely repopulates the aged livers over time; the number of periportal positive LRP1+ HEPs increases as the animals age. To determine if these LRP1+ and LRP1− HEPs are functionally different, we stained livers from aged animals for several different proteins and found that the hepatocellular transcription factor, HNF4α, a master regulator of hepatic function, was specifically absent in the LRP1− HEPs where lipids are abundant. Upon further investigation, we determined that in WT animals, LRP1 and HNF4α can form a complex that is present in both the nucleus and cytoplasm, leading us to hypothesize that LRP1 is capable of trafficking HNF4α as a means of regulating lipid metabolism. To begin to investigate this hypothesis, we took advantage of the plasminogen activator inhibitor type 1 (PAI‐1) knockout (KO) mouse as PAI‐1 is known to regulate the internalization of LRP1.MethodsTo confirm complex formation, immunoprecipitation (IP) was performed using total cell lysates from livers of PAI‐1 wild type (WT) and KO mice that were fed either normal chow (NC) or high fat diet (HFD). Next, cytoplasmic‐ and nuclear‐enriched extracts were prepared from these livers and probed by western blot to determine if diet affected complex location. Finally, immunohistochemistry (IHC) was used to localize the proteins.ResultsOverall complex formation was increased in the animals on the HFD, regardless of genotype. Similarly, less complex was found in the cytoplasms of animals on HFD, relative to their NC counterparts, regardless of genotype. Finally, localization of HNF4α was found to be regionalized in the PAI‐1 KOs on HFD, but not in their WT counterparts; HNF4α was present in areas of fat deposition in the PAI‐1 WT animals, but absent in the KOs, further implicating LRP1 in HNF4α trafficking.ConclusionThese findings confirm that LRP1 and HNF4α can form a complex, that diet affects quantity and location of the complex, and further indicates that LRP1 has a role in regulating nuclear localization of HNF4α. Further studies will be needed to elucidate the exact function of the complex, as well as its possible implications for lipid regulation and metabolism.Support or Funding InformationSarah Hoskings was supported by an SROPP award from ASIP as well as SURP funding from the family of Dr. William Zeiler.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A complex between phosphatidylinositol 4-kinase IIα and integrin α3β1 is required for N-glycan sialylation in cancer cells

Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3β1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3β1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3β1.

Increasing the low lipid phosphate phosphatase 1 activity in breast cancer cells decreases transcription by AP-1 and expressions of matrix metalloproteinases and cyclin D1/D3.

Metastasis is the leading cause of mortality in breast cancer patients and lysophosphatidate (LPA) signaling promotes this process. LPA signaling is attenuated by lipid phosphate phosphatase-1 (LPP1) whose activity is decreased in cancers. Consequently, increasing LPP1 levels suppresses breast tumor growth and metastasis. This study shows that increasing LPP1 in breast cancer cells decreases transcription through cFos and cJun. This decreases production of cyclin D1/D3 and matrix metalloproteinases (MMPs), which provides new insights into the role of LPP1 in controlling tumor growth and metastasis.Methods: Invasiveness was determined by a Matrigel invasion assay. MMP expression was measured by qPCR, multiplex LASER bead technology and gelatin zymography. Levels of cJUN, cFOS, FRA1, cyclin D1, and cyclin D3 were determined by qPCR and western blotting. Collagen was determined by Picro-Sirius Red staining.Results: Increasing LPP1 expression inhibited invasion of MDA-MB-231 breast cancer cells through Matrigel. This was accompanied by decreases in expression of MMP-1, -3, -7, -9, -10, -12 and -13, which are transcriptionally regulated by the AP-1 complex. Increasing LPP1 attenuated the induction of mRNA of MMP-1, -3, cFOS, and cJUN by EGF or TNFα, but increased FRA1. LPP1 expression also decreased the induction of protein levels for cFOS and cJUN in nuclei and cytoplasmic fractions by EGF and TNFα. Protein levels of cyclin D1 and D3 were also decreased by LPP1. Although FRA1 in total cell lysates or cytoplasm was increased by LPP1, nuclear FRA1 was not affected. LPP1-induced decreases in MMPs in mouse tumors created with MDA-MB-231 cells were accompanied by increased collagen in the tumors and fewer lung metastases. Knockdown of LPP1 in MDA-MB-231 cells increased the protein levels of MMP-1 and -3. Human breast tumors also have lower levels of LPP1 and higher levels of cJUN, cFOS, MMP-1, -7, -8, -9, -12, -13, cyclin D1, and cyclin D3 relative to normal breast tissue.Conclusion: This study demonstrated that the low LPP1 expression in breast cancer cells is associated with high levels of cyclin D1/D3 and MMPs as a result of increased transcription by cFOS and cJUN. Increasing LPP1 expression provides a novel approach for decreasing transcription through AP-1, which could provide a strategy for decreasing tumor growth and metastasis.

Label-free Quantitative Phosphoproteomics Reveals the Role of Beta-Estradiol in Sunitinib-Resistant Renal Cell Carcinoma Growth Via Perturbing Transforming Growing Factor-Beta Pathway

Context: Sunitinib is the first-line targeted therapy for metastatic renal cell carcinoma (RCC). However, resistance to sunitinib often occurred in patients receiving sunitinib treatment. On the other hand, 17-beta-estradiol (estrogen or E2) has been demonstrated to repress RCC growth in vitro, whether E2 can also affect the growth of sunitinib-resistant RCC remains unknown. Aims: In this study, the role of E2 in inhibiting sunitinib-resistant RCC growth and the underlining acting mechanisms was explored. Settings and Design: Sunitinib resistance was first induced in vitro in ACHN cells. The effect of E2 on cellular growth was then assayed. Label-free phosphoproteomics was also conducted. Subjects and Methods: ACHN cells were first challenged with 10-μM sunitinib up to 4 months to induce drug resistance. Then, E2 at different concentrations were tested in both parental and sunitinib-resistant ACHN cells. To conduct phosphoproteomics study, the total cell lysates from E2-treated ACHN cells were harvested, trypsin digested, and the phosphopeptides were enriched by Fe-IMAC. Statistical Analysis Used: For comparing the E2-induced cell growth inhibition, Student's t-test was used, and P &lt; 0.05 was considered statistically significant. As for label-free phosphoproteomics, false discovery rate &lt;0.01 and phosphosite possibility &gt;0.75 were considered as positive identifications. Results: E2 at the physiological concentration, that is, 10 nM, can repress the cell growth in both parental and sunitinib-resistant ACHN cells. Further, label-free phosphoproteomics revealed that transforming growth factor beta (TGF-β) pathway, cell cycle, and cytoskeleton bindings were enhanced in sunitinib-resistant cells but can be reduced by E2 treatment. On the other hand, programmed cell death and apoptosis were repressed in sunitinib-resistant cells, and E2 at 10 nM did not reverse the effect. We further validated the expression of SMAD3, an important molecule in TGF-β pathway, and found that SMAD3 decreased in sunitinib-resistant cells but can be upregulated by E2 treatment. Conclusions: Our study demonstrated that E2 can inhibit the cell growth in sunitinib-resistant RCC cells at physiological concentration by upregulating SMAD3 in the TGF-β pathway, which may lead to growth inhibition in RCC.

Recombinant Antigens In Serological Diagnosis Of Lyme Borreliosis

Abstract Lyme borreliosis, an infectious disease caused by tick-borne spirochetes of the Borrelia burgdorferi sensu lato complex, is regarded as the most commonly reported vector-borne infection in the Northern Hemisphere. Currently, the basis for laboratory diagnosis of Lyme disease is a two-step serological examination. The first is an enzyme-linked immunosorbent assay (ELISA). If the test result is positive or questionable, a Western blot is used as the second phase test. In both methods, the total cell lysates of B. burgdorferi s.l. are used as the main source of antigens. However, the huge diversity of genospecies within B. burgdorferi s.l. and the low degree of preservation of the sequence of their proteins means that using the cell lysates of one of the species is not sufficient to correctly diagnose Lyme disease. Numerous literature reports show that the use of B. burgdorferi s.l. recombinant or chimeric antigens may be a potential solution to problems occurring in Lyme disease immunodiagnosis. However, for diagnostic tests based on recombinant proteins to be as effective as possible, carefully selected antigens or fragments should be used. With this approach, a test can be developed with a sensitivity that remains independent of the B. burgdorferi s.l. species which caused the disease. In addition, the exclusive use of protein fragments may definitely reduce the frequency of cross-reactions. 1. Introduction. 2. Characterization of selected B. burgdorferi s.l. antigens. 3. Diagnosis of Lyme disease. 4. Problems in Lyme disease serodiagnosis. 5. Use of recombinant antigens and synthetic peptides in the diagnosis of Lyme disease. 6. Summary

Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts.

ABSTRACTN-cadherin mediates cell–cell contacts in vascular smooth muscle cells (VSMCs), and regulates VSMC behaviours including migration and proliferation. Discoidin domain receptor 1 (DDR1) is a collagen binding receptor also implicated in these processes. Previous studies have shown that both N-cadherin and DDR1 are upregulated after vascular injury, but it is not known whether there is a relationship between the two molecules. In the current study we found that N-cadherin was mislocalised from cell–cell junctions in the absence of DDR1. This occurred in spite of the fact that there was no significant difference in total cell lysate levels of N-cadherin between DDR1+/+ and DDR1−/− VSMCs. Analysis of lipid raft fractions revealed decreased N-cadherin and associated junctional complex catenins in DDR1−/− compared to DDR1+/+ VSMCs. Treatment with cholesterol oxidase or methyl-β-cyclodextrin to disrupt lipid rafts removed N-cadherin and DDR1 from the raft fractions. Reciprocal co-immunoprecipitations suggested the association of DDR1 and N-cadherin. Importantly, transfection of DDR1−/− cells with full-length DDR1b rescued the formation of N-cadherin junctions. Together, these data reveal that N-cadherin cell–cell contacts in VSMCs are regulated through interactions with DDR1 and both molecules are located in lipid rafts.

Exploiting Substrate Promiscuity To Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes.

Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme's crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.

Defective Autophagy Process and Increased ROS Formation in Primary Myelofibrosis

Introduction: We and others have reported markedly increased ROS formation in primary myelofibrosis (MF) including post-ET or PV-MF when compared with essential thrombocytosis (ET), polycythemia vera (PV) and normal controls. A growing body of evidence illustrates the interdependency between the Keap1-Nrf2 pathway and p62-mediated selective autophagy, are playing an important role in major cellular defense mechanisms against oxidative and electrophilic stress ; dysregulation of p62-Keap1-Nrf2 axis has been implicated in tumor development. Therefore, we studied p62 mediated autophagy and Nrf2 pathways in patients with MF ,ET and PV.Materials and Methods:33 patients including 15 patients with MF, 7 patients with PV, 11 patients with ET and 10 normal controls were studied. Cellular ROS measurement: Cellular ROS was determined by a dichlorofluorescein (DCF) assay. The oxidation of H2DCFDA was measured by flow cytometry. Real-time PCR assay: Total RNA was extracted from normal control or patient mononuclear cells using RNeasy Kits from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Western blotting: Total cell lysates were obtained from normal control or patient mononuclear cells. Primary antibodies against Beclin-1, LC3 and p62 were purchased from Cell Signaling Technology (Danvers, MA).Results: Fig.1a shows significantly elevated ROS in MF (2127±261.6) when compared with PV (1090 ± 163.8) or ET (1031 ± 140.9) or controls (549.1 ± 52.50). Fig.1b. shows that Nrf2 protein levels (ratio to controls) was significantly reduced in MF (41.84 ± 29.33), PV (14.42 ± 2.96), ET (14.05 ± 3.02), than the controls ( 101.3 ± 7.64). Fig.1c shows that the RT-PCR values of p62 was significantly increased in MF (1.34 ± 0.06) and grouped PV (1.21 ± 0.07), ET (1.14 ± 0.08) and MF as MPN (1.27 ± 0.04) than controls (1.00 ± 0.05),( P<0.05); ET or PV were not significantly elevated than controls.Conclusions: Patients with MF had increased levels of ROS when compared with PV and ET while Nrf2 levels were significantly reduced in all three( ET,PV, MF) which implies defective detoxification process in MPNs. P62, a marker for defective autophagy was significantly increased in MF than controls . This implies that there is a defective autophagy process in MF, contributing further to the formation of P62 and ROS. There is also a defective detoxification mechanism which further leads to more ROS formation and further DNA damage. Further studies are in progress on other autophagy markers which will enlighten more about the pathophysiology of MF. [Display omitted] DisclosuresWang:INCYTE: Other: clinical trial.

PO-119 Adrenergic Receptor β3 Up-regulates Uncoupling Protein 1 and Cyclooxygenase 2 Expressions in The Brown Adipocyte

Objective Brown adipose tissues (BAT) activation is important for losing weight as its high energy expenditure in Mammalian. Recent studies showed that exercise may also be essential for BAT activation. Uncoupling protein 1 (UCP1), specifically expressed in BAT's mitochondria, uncouples oxidative phosphorylation and dissipates energy from Free Fatty Acids into heat. Activating the Adrenergic Receptor β3 (Adrβ3) provides fuel for mitochondrial heat production and up-regulates Cyclooxygenase 2 (COX2), which is a key factor of UCP1 synthesis. Sympathetic nerve excitement stimulated by exercise can release norepinephrine as a neurotransmitter, which can affect Adrβ3. Brown adipocyte (BAC) is a kind of adipocyte in vitro as a model to study heat production. Isoprenaline Hydrochloride (ISO) is a widely used as an Adrβ agonist. In this research, we tried to figure out the response of BAC to Adrβ3 activations with different time points and whether ISO can be used as a BAC activator.&#x0D; Methods C3H10T1/2 cells were maintained in a humidified, 37°C, 5% CO2 incubator in DMEM/F12 medium with 10% fetal bovine serum (FBS). For brown adipogenesis, cells were first split into differentiation medium (DMEM/F12 containing 10% FBS, 20nM insulin, 1nM 3,3’5-Triiodo-L-thyronine(T3)) for 4 days, the medium was changed every other day. Confluent cells were treated for 2 days with brown adipose adipogenesis cocktails (differentiation medium containing 2µg/mL dexamethasone, 0.5mM isobutylmethylxanthine (IBMX), 0.125mM indomethacin and 1µM rosiglitazone) on day 4. Then the medium was replaced by differentiation medium and changed every other day. At day 10, the full differentiation adipocytes were treated with 10µM ISO for 0 (as control), 1, 3, 6, 12 and 24 hours. For the lipid droplets staining, the cells were fixed by 4% paraformaldehyde solution then stained with Oil Red O. The cells were harvested and the total cell lysates were extracted for protein analysis after each time point. The UCP1, COX2, and Adrβ3 expression levels were detected by western blot, using Actin as the internal protein. The results were expressed as the mean ± standard error of the mean (SEM). Group comparisons were performed using two-way ANOVA and LSD’s post-hoc tests.&#x0D; Results After differentiation, the cell shapes converted from fibroblastic to a spherical shape. Dispersed small lipid droplets were observed in the cells. After ISO treatment, the red color after Oil Red Staining became lighter and the size of the lipid droplets turned to smaller. The Adrβ3 protein expressions were 1.00±0.00, 1.34±0.32, 1.07±0.50, 4.65±1.84*, 2.44±0.73, and 3.43±1.09 at 0h, 1h, 3h, 6h, 12h, and 24h after ISO treatment, respectively. After introduced to ISO, the UCP1 expression levels were 1.00±0.00, 1.95±0.39, 2.72±0.57, 5.68±1.82*, 3.49±0.92, and 2.79±1.05 at 0h, 1h, 3h, 6h, 12h, and 24h, respectively. And for COX2, the protein expressions were 1.00±0.00, 2.13±0.67, 1.82±0.33, 4.67±1.82*, 2.88±0.44, and 2.65±0.54, respectively. The * means p ˂ 0.05, compared with oh controls. The proteins expressions were reached to peak after 6 hours ISO treatment from the above results.&#x0D; Conclusions UCP1 and COX2 protein expressions were increased in BAC according to Adrβ3's expression in different time points, indicating that Adrβ3 may induce adipolysis in BAC and help to burn fat and produce heat.

Identification of targets of monoclonal antibodies that inhibit adhesion and growth in Mycoplasma mycoides subspecies mycoides

Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.