Browsing by Subject "Cell cycle"

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    Functional characterization of the three isoforms of Fbw7 (F-box and WD repeat domain containing 7) in ubiquitin dependent proteolysis.
    (2010-01) Zhang, Wei
    Fbw7 is the F-box protein of SCFFbw7 E3 ubiquitin ligase, which specifically associates with the substrates to be ubiquitinated. Substrates of Fbw7 play important roles in cell cycle regulation, proliferation, signal transduction and metabolism, which are related to tumor formation, suggesting that Fbw7 functions as a tumor suppressor. Fbw7 has three splicing variants α, β and γ, and the biological function of each isoform is not well understood. Our lab is interested in how the Fbw7 isoforms regulate cyclin E proteolysis and the cell cycle. By using mammalian and insect cell culture systems, I demonstrate that the three isoforms can form homo- and heterodimers in vivo and in vitro. The dimerization domain is located immediately upstream of the F-box motif, and it is highly conserved in all Fbw7 homologues and other related F-box proteins, indicating the dimerization may be common feature of a subset of F-box proteins. Abolishment of dimerization inhibits cyclin E proteolysis and leads to a prolonged half-life of cyclin E, although it does not affect Fbw7 binding to cyclin E or to the Cul-Rbx1-Skp1 E3 catalytic module. Cyclin E accumulation can be commonly found in many primary tumors and cancer cell lines. These results suggest a novel mechanism of how F-box proteins recognize their substrates. Fbw7 isoforms show different protein stabilities, where the α isoform is stable, but the β and γ isoforms are not. The stability of the β and γ isoforms is largely controlled by their N- terminal unique region. In order to better understand the mechanism regulating their stability, we performed a yeast two hybrid screen and identified SLP1 (stomatin like protein 1) as an Fbw7γ isoform specific interacting protein. SLP1 binds to the unique region of γ isoform, and stabilizes γ. We find that Cdk2 promotes the degradation of both SLP1 and the γ isoform, and this function of Cdk2 is dependent on its kinase activity. SLP1 also physically interacts with Cdk2 through its membrane association domain. These results support a model in which Fbw7γ and SLP1 are coordinately targeted for ubiquitin mediated degradation by Cdk2.
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    Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis.
    (2011-07) Khammanivong, Ali
    Malignant transformation in squamous cell carcinomas (SCC) such as those of the head and neck (HNSCC) remains an enigmatic process that results in abnormal cellular differentiation, loss of growth and cell cycle regulation, gain of replicative immortality and resistance to apoptosis, activation of cellular migration and invasion, increase in energy metabolism, and evasion of immune destruction. These abnormalities in cell functions emerged as the hallmarks of cancer. Calprotectin, a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex (EDC), may play essential roles in the regulation of cell differentiation, cell cycle progression, cellular survival and cell migration that are part of the cancer hallmarks. While highly upregulated in a variety of cancers, calprotectin is down-regulated in squamous cell carcinomas of the cervix, esophagus and the head and neck. Using microarray analysis for gene expression we found that the S100A9 subunit of calprotectin was significantly down-regulated along with other EDC genes in human primary HNSCC cases, suggesting a loss of functional S100A8/A9 protein complex and differentiation during carcinogenesis. Expression of S100A9 correlated strongly with a set of HNSCC downregulated genes putatively involved in loss of cytodifferentiation and control of cell cycle. To probe its role in carcinogenesis, S100A8/A9 was stably expressed in a calprotectin-negative human carcinoma cell line (KB cells). Expression of S100A8/A9 in KB cells up-regulated differentiation and cell-cell contact growth inhibition signaling pathways, re-established epithelial actin microfilament cytoskeletal structures and v cellular adhesion to the extracellular matrix, down-regulated anti-apoptosis gene networks, and suppressed anchorage-independent survival and stress-induced (by serum starvation) cell migration. Calprotectin appeared to induce growth suppression by signaling a reactivation of G2/M cell cycle checkpoint regulators, Chk1 and PP2A, and inactivation of mitotic activators Cdc25C and Cdc2. As a result, we saw a marked increase in Cdc2-PThr14/Tyr15 phosphorylation and down-regulation of cyclin B1, suggesting an inactivation of the mitotic entry promoting Cdc2/cyclin B1 complex, resulting in cell cycle and mitotic arrest at the G2/M checkpoint. When inoculated into nude mice, KB cells producing calprotectin showed reduced tumor growth when compared to sham-transfected control KB cells. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increased carcinoma growth and survival and reduced Cdc2 phosphorylation at Thr14/Tyr15. Calprotectin expression in KB and TR146 cells also down-regulated expression of putative HNSCC marker genes, INHBA, PTGS2 (Cox-2) and SULF1, found to be expressed only in HNSCC samples. Calprotectin-mediated control of cellular differentiation and G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may point to new molecular targets for therapy.