Ubiquitination plays a crucial role in regulating protein turnover. Here we show that ubiquitination regulates the stability of the MDR1 gene product, P-glycoprotein, thereby affecting the functions of this membrane transporter that mediates multidrug resistance. We found that P-glycoprotein was constitutively ubiquitinated in drug-resistant cancer cells. Transfection of multidrug-resistant cells with wild-type ubiquitin or treatment with an N-glycosylation inhibitor increased the ubiquitination of P-glycoprotein and increased P-glycoprotein degradation. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), a proteasome inhibitor, induced accumulation of ubiquitinated P-glycoprotein, suggesting the involvement of the proteasome in the turnover of the transporter. Treatment of multidrug-resistant cells with 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that increases the phosphorylation of P-glycoprotein through activation of protein kinase C, or substituting phosphorylation sites of P-glycoprotein by nonphosphorylatable residues did not affect the ubiquitination of the transporter. Enhanced ubiquitination of P-glycoprotein resulted in a decrease of the function of the transporter, as demonstrated by increased intracellular drug accumulation and increased cellular sensitivity to drugs transported by P-glycoprotein. Our results indicate that the stability and function of P-glycoprotein can be regulated by the ubiquitin-proteasome pathway and suggest that modulating the ubiquitination of P-glycoprotein might be a novel approach to the reversal of drug resistance.Drug resistance is a major impediment to successful cancer chemotherapy. Multidrug resistance (MDR) mediated by Pglycoprotein (P-gp), the product of the MDR1 (ABCB1) gene, is believed to be one of the major causes of failure of cancer therapy. P-gp is a 150-to 180-kDa heavily glycosylated and phosphorylated plasma membrane protein that functions as a drug transporter. Overexpression of P-gp confers resistance to a variety of structurally and functionally diverse anticancer drugs such as paclitaxel, doxorubicin, and vinblastine. Despite promising early studies showing that blocking of P-gp by pharmacological means could sensitize drug-resistant cells, the ultimate goal of restoring drug sensitivity has met with limited success in clinical trials. Therefore, we and others began to elucidate the factors that control P-gp synthesis. For example, we have demonstrated that several substances control the expression of MDR1 through activation of phospholipase C and that the transcriptional modulation of MDR1 expression by phospholipase C is mediated by the Raf-mitogen-activated protein kinase pathway (Yang et al., 2001). We recently have become interested in the factors that regulate P-gp stability.P-gp, at steady state, is located in the plasma membrane and undergoes endocytosis and recycling (Kim et al., 1997). Experimentally induced alterations in trafficking of P-gp can change the steady-state distribution of the transporter, thereby affecting the...

SUMMARYNumerical simulation of cracked structures is an important aspect in structural safety assessment. In recent years, there has been an increasing rate of development of numerical codes for modelling fracture procedure. The subject of this investigation is implementing automated fracture models in the DYNA3D non-linear explicit ÿnite element code to simulate pseudo 3D crack growth procedure. The implemented models have the capabilities of simulating automatic crack propagation without user intervention. The implementation is carried on solid elements. The methodology of implementing fracture models is described. An element deletion-and-replacement remeshing procedure is proposed for updating the explicit geometric description of evolving cracks. Fracture parameters such as stress intensity factors, energy release rates and crack tip opening angle are evaluated. The maximum circumferential stress criterion is used to predict the direction of crack advancement. Seven crack problems are presented to verify the e ectiveness of the methodology. Mesh sensitivity and loading rate e ects are studied in the validation of the presented procedure.

ABSTRACT:The subject of this investigation is the development of an accurate simulation of a truck impacting a strong-post w-beam guardrail. Detailed methods for system simulation are proposed and three major issues which involve the use of springs to simulate component crashworthiness behavior is investigated. Rail to blockout bolt connection, soil-post dynamic interaction, and effect of ends of guardrail are modeled and simulated. Soilpost interaction is modeled using both Lagrangian and Eulerian meshes and results using the two methods are presented. The present paper provides a roadmap for simulation of highway safety structures.

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