Biomedical subjects

Eileen Turcott

Publications and source records attributed to Eileen Turcott.

5 recordsLinked to original sources

PubMed2006

Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment.

The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.

Animals↗

PubMed2004

Sclerostin inhibition of Wnt-3a-induced C3H10T1/2 cell differentiation is indirect and mediated by bone morphogenetic proteins.

High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone.

The transcriptome provides the database from which a cell assembles its collection of proteins. Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels. Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery. Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels. Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone. Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies. These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions.

Computational Biology↗

PubMed2004

Sclerostin promotes the apoptosis of human osteoblastic cells: a novel regulation of bone formation.

A null mutation in the SOST gene is associated with sclerosteosis, an inherited disorder characterized by a high bone mass phenotype. The protein product of the SOST gene, sclerostin, is a bone morphogenetic protein (BMP) antagonist that decreases osteoblast activity and reduces the differentiation of osteoprogenitors. We sought to delineate the mechanism by which sclerostin modulated osteoblastic function by examining the effects of the protein on differentiating cultures of human mesenchymal stem cells (hMSC). Sclerostin significantly decreased alkaline phosphatase (ALP) activity and the proliferation of hMSC cells. In addition, hMSC cells treated with sclerostin displayed a marked increase in caspase activity. Elevated levels of fragmented histone-associated DNA in these cells were detected by ELISA and by TUNEL staining. Other BMP antagonists including noggin, Chordin, Gremlin, and Twisted gastrulation did not affect caspase activity. The sclerostin-mediated increase in caspase activity was blocked by caspase-1 and caspase-3 inhibitors. Sclerostin-induced changes in ALP activity and the survival of hMSC cells were partially restored by BMP-6, suggesting the involvement of additional growth factors. These findings show that sclerostin selectively controls the apoptosis of bone cells. The ability of sclerostin to interact with important growth factors such as BMPs likely serves as the basis by which it modulates the survival of osteoblasts. By making these growth factors unavailable for cell function, sclerostin promotes the apoptosis of bone cells, providing a novel level of control in the regulation of bone formation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The two upstream open reading frames of oncogene mdm2 have different translational regulatory properties.

Few details are known of the mechanisms through which multiple upstream open reading frames (uORFs) interact to regulate translation in higher eukaryotes. The predominant transcript of oncogene mdm2 in normal human cells (L-mdm2) contains two upstream open reading frames in its 5' leader. Elimination of these two uORFs raises the translational efficiency of the transcript by over 10-fold in HeLa cells. The 5'-most uORF (uORF1) alone suppresses downstream translational activity by over 5-fold, whereas uORF2 contributes <2-fold to the inhibition by the intact leader. The different activities of the two uORFs do not depend on the nucleotide sequence surrounding the uORFs in the 5' leader, the order of the two uORFs in the 5' leader, or the occurrence of secondary structure or rare codons within the uORFs. Specific features of the amino acid sequence encoded by uORF1 contribute to its stronger suppressive activity, suggesting that it belongs to the class of "sequence-specific" uORFs. The weaker inhibitory activity inherent in uORF2 is potentiated by a sub-optimal nucleotide context surrounding its initiator AUG. The occurrence of two uORFs with differing activities in both the human gene and the mouse orthologue suggests that this pair of elements may play a fundamental role in regulating expression of the mdm2 gene.

5' Untranslated Regions↗