Calreticulin: non-endoplasmic reticulum functions in physiology and disease.

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.

LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies.

The LDL receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes, and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, macrophages, and adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: 1) its ability to recognize more than 30 distinct ligands, 2) its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner, and 3) its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases.

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells.

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl(-) channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V(Post-RVD) < V(baseline)). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl(-) channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd(2+) (V(Post-RVD) > 1.4*V(baseline)). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K(+)-Cl(-) cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15 degrees C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15 degrees C, the channel blockers NPPB + Cd(2+) completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl(-) channels vs. transporters to this process is approximately 60-70% and approximately 30-40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl(-) channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a.

Phase II trial of gefitinib in recurrent glioblastoma.

PURPOSE: To evaluate the efficacy and tolerability of gefitinib (ZD1839, Iressa; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent glioblastoma. PATIENTS AND METHODS: This was an open-label, single-center phase II trial. Fifty-seven patients with first recurrence of a glioblastoma who were previously treated with surgical resection, radiation, and usually chemotherapy underwent an open biopsy or resection at evaluation for confirmation of tumor recurrence. Each patient initially received 500 mg of gefitinib orally once daily; dose escalation to 750 mg then 1,000 mg, if a patient received enzyme-inducing antiepileptic drugs or dexamethasone, was allowed within each patient. RESULTS: Although no objective tumor responses were seen among the 53 assessable patients, only 21% of patients (11 of 53 patients) had measurable disease at treatment initiation. Seventeen percent of patients (nine of 53 patients) underwent at least six 4-week cycles, and the 6-month event-free survival (EFS) was 13% (seven of 53 patients). The median EFS time was 8.1 weeks, and the median overall survival (OS) time from treatment initiation was 39.4 weeks. Adverse events were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent at higher doses. Withdrawal caused by drug-related adverse events occurred in 6% of patients (three of 53 patients). Although the presence of diarrhea positively predicted favorable OS from treatment initiation, epidermal growth factor receptor expression did not correlate with either EFS or OS. CONCLUSION: Gefitinib is well tolerated and has activity in patients with recurrent glioblastoma. Further study of this agent at higher doses is warranted.