Biomedical subjects

Elayne Provost

Publications and source records attributed to Elayne Provost.

6 recordsLinked to original sources

PubMed2006

The Suppressor of Killer of prune, a unique glutathione S-transferase.

The prune-Killer of prune conditional dominant, lethal interaction in Drosophila was identified in the 1950s, but its mechanism remains unknown. We undertook a genetic screen for suppressors of this lethal interaction and identified a gene we named, Suppressor of Killer of prune Su(Kpn). Su(Kpn) is a unique protein with four N-terminal FLYWCH zinc-finger domains, an acidic domain and a C-terminal glutathione S-transferase (GST) domain. The GST domain of Su(Kpn) is of particular interest because GSTs are usually independent of other protein domains. While GSTs are generally thought of as detoxifying enzymes, they are also associated with cellular toxicity. We predict that the GST domain of the Su(Kpn) creates a toxic product in prune-Killer of prune flies that is lethal. The substrate of the Su(Kpn) remains unknown.

Animals↗

PubMed2005

Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.

The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity.

Animals↗

PubMed2005

Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin.

Beta-catenin is a multifunctional protein involved in both cadherin-mediated adhesion and the wnt signaling cascade. Mutations in exon 3 of beta-catenin have been identified in many cancers. In addition to disruption of key serine and threonine residues, mutations are frequently reported in other residues in exon 3 that are not kinase substrates. The most frequently mutated nonserine/threonine residues are D32 and G34. Since D32 and G34 are part of the ubiquitination destruction motif, DSGPhiXS, we hypothesize that this motif may contribute to disruption of beta-catenin homeostasis and lead to cellular transformation. We demonstrate that the mutants D32A and G34A exhibit no change in phosphorylation by GSK3beta, but display reduced ubiquitination compared to wild-type and S33A mutant beta-catenin. To assess the functional implications of these mutations, we created stable MDCK cell lines expressing these constructs. We found that stable cell lines harboring D32A-mutated beta-catenin were highly transformed, while S33A and G34 demonstrated only weak transforming properties in our assays. Despite altered ubiquitination status and increased transformation, the D32A mutant cell line does not display transcriptional activation of standard target genes. Therefore, D32A mutation may mediate transformation by an alternative beta-catenin-mediated signaling pathway.

Aspartic Acid↗

PubMed2005

beta-Catenin functions mainly as an adhesion molecule in patients with squamous cell cancer of the head and neck.

BACKGROUND: beta-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of beta-catenin in head and neck carcinogenesis. METHODS: First, we studied the incidence of mutations of beta-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of beta-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data. RESULTS: The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. beta-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of beta-catenin (P = 0.040). Patients with low beta-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high beta-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low beta-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low beta-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only beta-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high beta-catenin had low cyclin D1 and vice versa (P = 0.007). CONCLUSIONS: The absence of activating beta-catenin mutations combined with the inverse correlation between beta-catenin levels with cyclin D1 levels and outcome suggest that beta-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Adult↗

PubMed2003

Functional correlates of mutations in beta-catenin exon 3 phosphorylation sites.

beta-Catenin-mediated signaling can be constitutively activated by truncation or mutation of serine and threonine residues in exon 3. Mutations in this region are observed in many human tumors. Examination of the locations of these mutations reveals interesting patterns; specifically, Ser45 and Thr41 appear more frequently in malignant tumors, and Ser37 and Ser33 are more common in benign entities. To test whether these patterns represent functional differences in beta-catenin signaling mechanisms, we generated mutations of each of these residues. Stable transformation of Madin-Darby canine kidney cells showed a transformed phenotype with each of the four mutations, as assessed by growth in soft agar and collagen. Functional assays including proliferation assays, cell shedding assays, and wounding assays demonstrated two groups. Ser45 and Thr41 represent a more transformed phenotype, whereas Ser37 and Ser33 behaved similarly to the vector in these assays. Assessment of downstream genes demonstrated increased activation of the beta-catenin target gene cyclin D1 by Ser45. Finally, we examined the kinase activity of I kappa B kinase-alpha and found that this kinase, unlike glycogen synthase kinase-3 beta, appears to preferentially phosphorylate Ser45 and Thr41, independent of priming by casein kinase-1. We conclude that these sites may represent an alternative (non-wnt) signaling pathway, which may be inappropriately activated in tumors with mutations of these residues.

Animals↗

PubMed2003

Tissue microarray-based analysis shows phospho-beta-catenin expression in malignant melanoma is associated with poor outcome.

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the beta-catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of beta-catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated beta-catenin, as detected by a phospho-specific antibody, may be useful to predict outcome in melanoma. We use immuno-histochemical analysis of beta-catenin and phospho-beta-catenin, first to verify the specificity of the phospho-beta-catenin antibody and then to assay expression in a tissue microarray-based study. The subcellular localization of beta-catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho-beta-catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho-beta-catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho-beta-catenin was more common in metastatic lesions. High levels of nuclear phospho-beta-catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho-specific antibodies to beta-catenin define a unique subset of cases and that monitoring of phospho-beta-catenin expression may be useful for assessing prognosis in malignant melanoma.

Cell Nucleus↗