Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique.

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.

A three-point identity criteria tool for establishing product identity using icIEF method.

Product identity is one of the release testing requirements that needs to be established to ensure that there is no misidentification of drugs. Here, we demonstrated the challenges that can come across while establishing a product identity method for monoclonal antibody (mAb) and mAb-related products using icIEF method. A unique three-point identity criteria tool (visual comparison, pI of individual peaks and ΔpIs) was applied to distinguish mAb1 from the other in-house mAbs. A reduction approach followed by icIEF showed higher potential for establishing identity for mAb1 product as compared to native and enzymatic digestion approach. In general, icIEF method lacks specificity required to unequivocally establish the identity for mAbs, therefore, risk analysis is recommended before implementing icIEF as a stand-alone identity method for monoclonal antibodies.

ERK and AKT signaling drive MED1 overexpression in prostate cancer in association with elevated proliferation and tumorigenicity.

MED1 is a key coactivator of the androgen receptor (AR) and other signal-activated transcription factors. Whereas MED1 is overexpressed in prostate cancer cell lines and is thought to coactivate distinct target genes involved in cell-cycle progression and castration-resistant growth, the underlying mechanisms by which MED1 becomes overexpressed and its oncogenic role in clinical prostate cancer have remained unclear. Here, we report that MED1 is overexpressed in the epithelium of clinically localized human prostate cancer patients, which correlated with elevated cellular proliferation. In a Nkx3.1:Pten mutant mouse model of prostate cancer that recapitulates the human disease, MED1 protein levels were markedly elevated in the epithelium of both invasive and castration-resistant adenocarcinoma prostate tissues. Mechanistic evidence showed that hyperactivated ERK and/or AKT signaling pathways promoted MED1 overexpression in prostate cancer cells. Notably, ectopic MED1 overexpression in prostate cancer xenografts significantly promoted tumor growth in nude mice. Furthermore, MED1 expression in prostate cancer cells promoted the expression of a number of novel genes involved in inflammation, cell proliferation, and survival. Together, these findings suggest that elevated MED1 is a critical molecular event associated with prostate oncogenesis.

Thyroid hormone suppression of ß-amyloid precursor protein gene expression in the brain involves multiple epigenetic regulatory events.

Thyroid hormone (T3) suppresses cerebral gene expression of the ß-amyloid precursor protein (APP), an integral membrane protein that plays a key role in the onset and progression of Alzheimer's disease. However, the mechanisms by which T3 signaling pathways inhibit APP gene transcription in the brain remain unclear. By carrying out chromatin immunoprecipitation with neuroblastoma cells and primary rat brain tissue, we show for the first time that thyroid hormone receptors (TRs) directly bind at the APP gene in vivo at a promoter region containing a negative T3-response element. We further show that T3 treatment decreases both histone H3 acetylation and histone H3 lysine 4 methylation at the APP promoter and that chemical inhibitors of histone deacetylases and histone lysine demethylase abrogate T3-dependent APP silencing. Our findings thus suggest that TRs actively facilitate T3-dependent silencing of APP gene expression via the recruitment of distinct histone modifying enzymes associated with transcriptional repression.

Repression of cardiac phospholamban gene expression is mediated by thyroid hormone receptor-{alpha}1 and involves targeted covalent histone modifications.

Phospholamban (PLB) is a critical regulator of Ca(2+) cycling in heart muscle cells, and its gene expression is markedly down-regulated by T(3). Nonetheless, little is known about the molecular mechanisms of T(3)-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TRalpha1 and TRbeta1 subtypes, and exhibits T(3)-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TRalpha1, but not TRbeta1, is essential for T(3)-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T(3)-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TRalpha1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitation and in vitro binding assays show that TRalpha1 directly binds at the PLB core promoter region. Furthermore, addition of T(3) triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T(3)-dependent repression of PLB in cardiac myocytes is directly facilitated by TRalpha1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing.

Cyclin-dependent kinase 8 positively cooperates with Mediator to promote thyroid hormone receptor-dependent transcriptional activation.

Mediator is a multisubunit assemblage of proteins originally identified in humans as a coactivator bound to thyroid hormone receptors (TRs) and essential for thyroid hormone (T3)-dependent transcription. Cyclin-dependent kinase 8 (CDK8), cyclin C, MED12, and MED13 form a variably associated Mediator subcomplex (termed the CDK8 module) whose functional role in TR-dependent transcription remains unclear. Using in vitro and cellular approaches, we show here that Mediator complexes containing the CDK8 module are specifically recruited into preinitiation complexes at the TR target gene type I deiodinase (DioI) together with RNA polymerase II (Pol II) in a TR- and T3-dependent manner. We found that CDK8 is essential for robust T3-dependent Dio1 transcription and that CDK8 knockdown via RNA interference decreased Pol II occupancy, and also the recruitment of the Pol II kinase CDK9, at the DioI promoter. Chromatin immunoprecipitation revealed CDK8 occupancy at the DioI promoter concurrent with active transcription, thus suggesting CDK8 involvement in transcriptional reinitiation. Mutagenesis assays showed that CDK8 kinase activity is necessary for full T3-dependent DioI activation, whereas in vitro kinase studies indicated that CDK8 may contribute to Pol II phosphorylation. Collectively, our data suggest CDK8 plays an important coactivator role in TR-dependent transcription by promoting Pol II recruitment and activation at TR target gene promoters.

MED1 phosphorylation promotes its association with mediator: implications for nuclear receptor signaling.

Mediator is a conserved multisubunit complex that acts as a functional interface between regulatory transcription factors and the general RNA polymerase II initiation apparatus. MED1 is a pivotal component of the complex that binds to nuclear receptors and a broad array of other gene-specific activators. Paradoxically, MED1 is found in only a fraction of the total cellular Mediator complexes, and the mechanisms regulating its binding to the core complex remain unclear. Here, we report that phosphorylation of MED1 by mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) promotes its association with Mediator. We show that MED1 directly binds to the MED7 subunit and that ERK phosphorylation of MED1 enhances this interaction. Interestingly, we found that both thyroid and steroid hormones stimulate MED1 phosphorylation in vivo and that MED1 phosphorylation is required for its nuclear hormone receptor coactivator activity. Finally, we show that MED1 phosphorylation by ERK enhances thyroid hormone receptor-dependent transcription in vitro. Our findings suggest that ERK phosphorylation of MED1 is a regulatory mechanism that promotes MED1 association with Mediator and, as such, may facilitate a novel feed-forward action of nuclear hormones.

Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells.

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.

Regulation of Aurora-A kinase gene expression via GABP recruitment of TRAP220/MED1.

The TRAP/Mediator coactivator complex serves as a functional interface between DNA-bound transactivators and the RNA polymerase II-associated basal transcription apparatus. TRAP220/MED1 is a variably associated subunit of the complex that plays a specialized role in selectively targeting TRAP/Mediator to specific genes. Ablation of the Trap220/Med1 gene in mice impairs embryonic cell growth, yet the underlying mechanism is unknown. In this report, we identified distinct cell growth regulatory genes whose expression is affected by the loss of TRAP220/MED1 by RNA interference. Among the down-regulated genes revealed by cDNA microarray analyses, we identified Aurora-A, a centrosome kinase that plays a critical role in regulating M phase events and is frequently amplified in several types of cancer. In general, we found that TRAP220/MED1 expression is required for high basal levels of Aurora-A gene expression and that ectopic overexpression of TRAP220/MED1 coactivates transcription from the Aurora-A gene promoter. Furthermore, chromatin immunoprecipitation assays show that TRAP220/MED1-containing TRAP/Mediator complexes directly bind to the Aurora-A promoter in vivo. Finally, we present evidence suggesting that TRAP/Mediator is recruited to the Aurora-A gene via direct interactions between TRAP220/MED1 and the Ets-related transcription factor GABP. Taken together, these findings suggest that TRAP220/MED1 plays a novel coregulatory role in facilitating the recruitment of TRAP/Mediator to specific target genes involved in growth and cell cycle progression.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.

Mechanism of inhibition of ascites tumor growth in mice by curcumin is mediated by NF-kB and caspase activated DNase.

One of the most clinically relevant biological activities of curcumin is its anti-cancer property, implicating multiple intracellular pathways in the process. In the present report, we investigated the effect of curcumin on the activation of apoptotic and anti-angiogenic pathways in Ehrlich Ascites Tumor (EAT) cells. Treatment with curcumin in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that the induction of apoptosis in EAT cells showed nuclear condensation, DNA fragmentation and translocation of caspase-activated DNase (CAD) to nucleus upon curcumin treatment. Curcumin-induced apoptosis is mediated through activation of caspase-3, which is specifically inhibited by the caspase-3 inhibitor, Ac-DEVD-CHO. On the other hand, the decreased secretion of ascites by EAT cells is corroborated by reduction in VEGF secretion upon curcumin treatment. Further, CD31 immunohistological staining of peritoneum sections in curcumin-treated mice suggests its efficacy in acting as anti-angiogenic compound in EAT cells by inhibiting proliferation of endothelial cells in mouse peritoneum. However, immunoflurescence studies of NF-kB revealed that the inhibition of nuclear translocation of NF-kB p65, a transcription factor required for VEGF gene expression, in curcumin-treated EAT cells. These results suggest a further possible clinical application of this diet-derived compound curcumin, as both proapoptotic and anti-angiogenic compound in association with conventional chemotherapeutic agents.

Antiangiogenic effects of butyric acid involve inhibition of VEGF/KDR gene expression and endothelial cell proliferation.

The formation of new blood vessels from pre-existing ones is required for the growth of solid tumors and for metastasis. Interaction of tumor-secreted vascular endothelial growth factor (VEGF) with its receptor(s) on endothelial cells triggers endothelial cell proliferation and migration, which facilitate tumor angiogenesis. Butyric acid (BuA), a fermentation product of dietary fibers in the colon, is shown to alter gene expression and is postulated to be anticarcinogenic. The results presented in this paper indicate that BuA can be antiangiogenic in vivo by inhibiting angiogenesis in chorioallantoic membrane assay. BuA was not cytotoxic to endothelial cells but was a potent antiproliferative agent besides being proapoptotic to endothelial cells as verified by FACS analysis. Conditioned media from BuA-treated Ehrlich ascites tumor cells showed a 30% decrease in VEGF concentration when compared with untreated cells. The decrease in VEGF mRNA and its receptor, KDR mRNA levels in EAT and endothelial cells respectively, suggests that the VEGF-KDR system of angiogenesis is the molecular target for the antiangiogenic action of BuA.

Molecular mechanisms of anti-angiogenic effect of curcumin.

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.