ABSTRACT: Human filamin C (FLNc) is a target of the protease calpain at Y2625. To confirm the dependency of calpain cleavage from FLNc phosphorylation at position S2623/S2624 by PKCa, FLNc was overexpressed in HEK293 cells and cells were treated with PMA to stimulated kinase activity, with Gö6976 to inhibit kinase activity or mock-treated with DMSO. Subsequently, recombinant calpain-1 and GluC were used and the resulting peptide TVTSSSSRGSSY was monitored by SRM analyses.
Project description:Human filamin C (FLNc) is a target of the protease calpain at Y2625. To confirm the dependency of calpain cleavage from FLNc phosphorylation at position S2623/S2624 by PKCa, FLNc was overexpressed in HEK293 cells and cells were treated with PMA to stimulated kinase activity, with Gö6976 to inhibit kinase activity or mock-treated with DMSO. Subsequently, recombinant calpain-1 and GluC were used and the resulting peptide TVTSSSSRGSSY was monitored by SRM analyses.
Project description:Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R(2) = 0.84) and urine (R(2) = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.
Project description:Gaussia luciferase (Gluc) is a sensitive reporter for studying different biological processes such as gene expression, promoter activity, protein-protein interactions, signal transduction, as well as tumor cell growth and response to therapy. Since Gluc is naturally secreted, the kinetics of these processes can be monitored in real-time by measuring an aliquot of conditioned medium in culture or a few microliters of blood in vivo at different time points. Gluc catalyzes light emission with a short half-life which is unfavorable for certain applications. We isolated a Gluc mutant that catalyzes enhanced light stability in the presence of a detergent, in combination with high sensitivity, making it an attractive luciferase for high-throughput functional screening applications.
Project description:Hydrogen sulfide is a toxic and corrosive gas, produced by the activity of sulfate-reducing microorganisms (SRM). Owing to the environmental, economic and human-health consequences of sulfide, there is interest in developing specific inhibitors of SRM. Recent studies have identified perchlorate as a promising emerging inhibitor. The aim of this work is to quantitatively dissect the inhibitory dynamics of perchlorate. Sulfidogenic mixed continuous-flow systems were treated with perchlorate. SRM number, sulfide production and community structure were monitored pre-, during and post-treatment. The data generated was compared to a simple mathematical model, where SRM growth slows as a result of inhibition. The experimental data supports the interpretation that perchlorate largely acts to suppress SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Surface-attachment was identified as an important parameter preventing SRM wash-out and thus governing inhibitory dynamics. Our study confirmed the lesser depletion of surface-attached SRM as compared to planktonic SRM during perchlorate treatment. Indirect effects of perchlorate (bio-competitive exclusion of SRM by dissimilatory perchlorate-reducing bacteria, DPRB) were also assayed by amending reactors with DPRB. Indeed, low concentrations of perchlorate coupled with DRPB amendment can drive sulfide concentrations to zero. Further, inhibition in a complex community was compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Finally, we quantified susceptibility to perchlorate across SRM in various culture conditions, showing that prediction of complex behavior in continuous systems from batch results is possible. This study thus provides an overview of the sensitivity of sulfidogenic communities to perchlorate, as well as mechanisms underlying these patterns.
Project description:BACKGROUND:Crizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non-small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue. METHODS:After validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib. RESULTS:We detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry. CONCLUSIONS:ALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies.
Project description:Raloxifene is a second-generation selective estrogen receptor modulator used for the prevention and treatment of osteoporosis and the prevention of breast cancer in postmenopausal women. Raloxifene is extensively metabolized by glucuronidation to form raloxifene-6-glucuronide (ral-6-Gluc) and raloxifene-4'-glucuronide (ral-4'-Gluc). The goal of the present study was to determine whether functional polymorphisms in active UGTs could play a role in altered raloxifene glucuronidation in vivo. Using homogenates from HEK293 UGT-overexpressing cell lines, raloxifene was shown to be glucuronidated primarily by the hepatic UGTs 1A1 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10; no detectable raloxifene glucuronidation activity was found for UGT2B enzymes. Functional UGT1A1 transcriptional promoter genotypes were significantly (Ptrend = 0.005) associated with ral-6-Gluc formation in human liver microsomes, and, consistent with the decreased raloxifene glucuronidation activities observed in vitro with cell lines overexpressing UGT1A8 variants, the UGT1A8*2 variant was significantly (P = 0.023) correlated with total raloxifene glucuronide formation in human jejunum homogenates. While ral-4'-Gluc exhibited 1:100th the anti-estrogenic activity of raloxifene itself as measured by binding to the estrogen receptor, raloxifene glucuronides comprised about 99% of the circulating raloxifene dose in raloxifene-treated subjects, with ral-4'-Gluc comprising ~70% of raloxifene glucuronides. Plasma ral-6-Gluc (Ptrend = 0.0025), ral-4'-Gluc (Ptrend = 0.001), and total raloxifene glucuronides (Ptrend = 0.001) were increased in raloxifene-treated subjects who were predicted slow metabolizers [UGT1A8 (*1/*3)] versus intermediate metabolizers [UGT1A8 (*1/*1) or UGT1A8 (*1/*2)] versus fast metabolizers [UGT1A8 (*2/*2). These data suggest that raloxifene metabolism may be dependent on UGT1A8 genotype and that UGT1A8 genotype may play an important role in overall response to raloxifene.
Project description:The carcinogenic effects of individual polycyclic aromatic hydrocarbons (PAH) are well established. However, their potency within an environmental complex mixture is uncertain. We evaluated the influence of diesel exhaust particulate matter on PAH-induced cytochrome P450 (CYP) activity, PAH-DNA adduct formation, expression of certain candidate genes and the frequency of tumor initiation in the two-stage Sencar mouse model. To this end, we monitored the effects of treatment of mice with diesel exhaust, benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP), or a combination of diesel exhaust with either carcinogenic PAH. The applied diesel particulate matter (SRM(1975)) altered the tumor initiating potency of DBP: a statistically significant decrease in overall tumor and carcinoma burden was observed following 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with DBP exposure alone. From those mice that were treated at the beginning of the observation period with 2 nmol DBP all survivors developed tumors (9 out of 9 animals, 100%). Among all tumors counted at the end, nine carcinomas were detected and an overall tumor incidence of 2.6 tumors per tumor-bearing animal (TBA) was determined. By contrast, co-treatment of DBP with 50mg SRM(1975) led to a tumor rate of only 66% (19 out of 29 animals), occurrence of only three carcinomas in 29 animals and an overall rate of 2.1 tumors per TBA (P=0.04). In contrast to the results with DBP, the tumor incidence induced by 200 nmol BP was found slightly increased when co-treatment with SRM(1975) occurred (71% vs. 85% after 25 weeks). Despite this difference in tumor incidence, the numbers of carcinomas and tumors per TBA did not differ statistically significant between both treatment groups possibly due to the small size of the BP treatment group. Since bioactivation of DBP, but not BP, predominantly depends on CYP1B1 enzyme activity, SRM(1975) affected PAH-induced carcinogenesis in an antagonistic manner when CYP1B1-mediated bioactivation was required. The explanation most likely lies in the much stronger inhibitory effects of certain PAHs present in diesel exhaust on CYP1B1 compared to CYP1A1. In the present study we also found molecular markers such as highly elevated AKR1C21 and TNFRSF21 gene expression levels in tumor tissue derived from animals co-treated with SRM(1975) plus DBP. Therefore we validate microarray data as a source to uncover transcriptional signatures that may provide insights into molecular pathways affected following exposure to environmental complex mixtures such as diesel exhaust particulates.
Project description:BACKGROUND & AIMS:Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS:A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS:Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS:Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.
Project description:Human promyelocytic leukaemia (HL-60) cells were employed to study the induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme in controlling prostaglandin inactivation. Phorbol 12-myristate 13-acetate (PMA) stimulated 15-PGDH activity in a time- and concentration-dependent manner. Dimethyl sulphoxide (DMSO) also stimulated the enzyme activity, although a much delayed stimulation was observed. Western blot studies indicated that PMA increased significantly a 28 kDa immunoreactive protein characteristic of 15-PGDH. L-[35S]Methionine labelling of the PMA-treated cells showed a similar enhancement over the control cells. These studies indicate that PMA induced synthesis of 15-PGDH. Stimulation of 15-PGDH activity by PMA or DMSO appears to be mediated by protein kinase C activation, since an inactive analogue of PMA failed to induce the effect, and both staurosporine and H-7 blocked the stimulation. Stimulation by PMA was optimal at 10 nM and less effective at higher concentrations. Western blot studies indicated that a similar, if not greater, amount of enzyme protein was induced at high concentrations of PMA, suggesting that enzyme inactivation might be occurring. Possible enzyme inactivation by protein kinase C activation was further examined by incubating DMSO-treated cells with a high concentration of PMA (50 nM). Time-dependent inactivation of 15-PGDH within the first 1 h was observed and this inactivation was partially blocked by staurosporine and H-7. Pulse-chase experiments indicated that 15-PGDH had a rapid turnover rate (t 1/2 = 47 min), and PMA shortened the half-life of the enzyme (t 1/2 = 33 min), suggesting that PMA might have an additional effect on 15-PGDH degradation. The rapid turnover of 15-PGDH indicates that the enzyme activity depends on continued enzyme synthesis, and this could be susceptible to hormone and drug control mechanisms.
Project description:Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.