Citations

Abstract Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. (Blood. 2003;102:4512-4519)

Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from–32.9±5.7 mV to +17.3±1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 μM ZnCl2(+43.0±4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4±1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C(PKC) δ-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCδ was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.

The apolipoprotein E ( APOE ) gene is the only genetic risk factor that has so far been linked to risk for late-onset Alzheimer's disease (LOAD). However, 50 percent of Alzheimer's disease cases do not carry an APOE4 allele, suggesting that other risk factors must exist. We performed a two-stage genome-wide screen in sibling pairs with LOAD to detect other susceptibility loci. Here we report evidence for an Alzheimer's disease locus on chromosome 10. Our stage one multipoint lod score (logarithm of the odds ratio for linkage/no linkage) of 2.48 (266 sibling pairs) increased to 3.83 in stage 2 (429 sibling pairs) close to D10S1225 (79 centimorgans). This locus modifies risk for Alzheimer's disease independent of APOE genotype.

Plasma Aβ42 (amyloid β42 peptide) is invariably elevated in early-onset familial Alzheimer's disease (AD), and it is also increased in the first-degree relatives of patients with typical late-onset AD (LOAD). To detect LOAD loci that increase Aβ42, we used plasma Aβ42 as a surrogate trait and performed linkage analysis on extended AD pedigrees identified through a LOAD patient with extremely high plasma Aβ. Here, we report linkage to chromosome 10 with a maximal lod score of 3.93 at 81 centimorgans close to D10S1225. Remarkably, linkage to the same region was obtained independently in a genome-wide screen of LOAD sibling pairs. These results provide strong evidence for a novel LOAD locus on chromosome 10 that acts to increase Aβ.

Abstract Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease caused by platelet destruction resulting from autoantibodies against platelet surface proteins, particularly platelet glycoprotein IIb/IIIa (αIIbβ3). To localize the auto-epitopes on platelet αIIbβ3, the binding of autoantibodies to Chinese hamster ovary (CHO) cells expressing either αIIbβ3 or αvβ3was studied. Thirteen of 14 ITP autoantibodies bound only to CHO cells expressing αIIbβ3. Because these 2 integrins have the same beta chain (β3), these results show that most epitopes in chronic ITP are dependent on the presence of glycoprotein αIIb.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Cross-sectional positron emission tomography (PET) studies find that cognitively normal carriers of the apolipoprotein E (APOE) ɛ4 allele, a common Alzheimer's susceptibility gene, have abnormally low measurements of the cerebral metabolic rate for glucose (CMRgl) in the same regions as patients with Alzheimer's dementia. In this article, we characterize longitudinal CMRgl declines in cognitively normal ɛ4 heterozygotes, estimate the power of PET to test the efficacy of treatments to attenuate these declines in 2 years, and consider how this paradigm could be used to efficiently test the potential of candidate therapies for the prevention of Alzheimer's disease. We studied 10 cognitively normal ɛ4 heterozygotes and 15 ɛ4 noncarriers 50–63 years of age with a reported family history of Alzheimer's dementia before and after an interval of approximately 2 years. The ɛ4 heterozygotes had significant CMRgl declines in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus, and these declines were significantly greater than those in the ɛ4 noncarriers. In testing candidate primary prevention therapies, we estimate that between 50 and 115 cognitively normal ɛ4 heterozygotes are needed per active and placebo treatment group to detect a 25% attenuation in these CMRgl declines with 80% power and P = 0.005 in 2 years. Assuming these CMRgl declines are related to the predisposition to Alzheimer's dementia, this study provides a paradigm for testing the potential of treatments to prevent the disorder without having to study thousands of research subjects or wait many years to determine whether or when treated individuals develop symptoms.