Citations

Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer–deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.

PURPOSE: The gastrointestinal Intergroup studied postoperative adjuvant chemotherapy and radiation therapy in patients with T3/4 and N+ rectal cancer after potentially curative surgery to try to improve chemotherapy and to determine the risk of systemic and local failure. PATIENTS AND METHODS: All patients had a potentially curative surgical resection and were treated with two cycles of chemotherapy followed by chemoradiation therapy and two additional cycles of chemotherapy. Chemotherapy regimens were bolus fluorouracil (5-FU), 5-FU and leucovorin, 5-FU and levamisole, and 5-FU, leucovorin, and levamisole. Pelvic irradiation was given to a dose of 45 Gy to the whole pelvis and a boost to 50.4 to 54 Gy. RESULTS: One thousand six hundred ninety-five patients were entered and fully assessable, with a median follow-up of 7.4 years. There was no difference in overall survival (OS) or disease-free survival (DFS) by drug regimen. DFS and OS decreased between years 5 and 7 (from 54% to 50% and 64% to 56%, respectively), although recurrence-free rates had only a small decrease. The local recurrence rate was 14% (9% in low-risk [T1 to N2+] and 18% in high-risk patients [T3N+, T4N]). Overall, 7-year survival rates were 70% and 45% for the low-risk and high-risk groups, respectively. Males had a poorer overall survival rate than females. CONCLUSION: There is no advantage to leucovorin- or levamisole-containing regimens over bolus 5-FU alone in the adjuvant treatment of rectal cancer when combined with irradiation. Local and distant recurrence rates are still high, especially in T3N+ and T4 patients, even with full adjuvant chemoradiation therapy.

PURPOSE: To evaluate the value of thymidylate synthase (TS), Ki-67, and p53 as prognostic markers in patients with Dukes’ B2 and C colon carcinoma. METHODS: We conducted a retrospective analysis to evaluate the prognostic value of TS, Ki-67, and p53 in 465 patients with Dukes’ B2 (220 patients) or Dukes’ C (245 patients) colon carcinoma. Patients represent a nonrandom subset obtained from five randomized phase III trials and were treated with either surgery alone (151 patients) or surgery plus fluorouracil-based chemotherapy (314 patients). All three markers were assayed using immunohistochemical techniques. RESULTS: With a minimum follow-up of 5 years, our retrospective analysis failed to demonstrate a consistent and significant association between TS, Ki-67, or p53 and either disease-free survival or overall survival. Exploratory analyses did not reveal a convincing explanation for these results that are in conflict with the published literature. Notable interactions were observed. In particular, high Ki-67 levels were associated with increased (decreased) survival in patients with low (high) TS intensity. Patients whose tumors stained positively for p53 seemed to benefit substantially from the use of adjuvant chemotherapy compared with those who were not treated (P = .05). CONCLUSION: This retrospective investigation failed to demonstrate a significant association between TS, Ki-67, or p53 staining and clinical outcome.

ABSTRACT An aspartyl protease, Beta‐Site APP cleaving enzyme (BACE), was identified as the β‐secretase responsible for generating the Amyloid β protein that is believed to cause Alzheimer's disease. BACE has a short propeptide domain that is absent in the mature enzyme because of proteolytic cleavage after the sequence RLPR. This sequence is a predicted substrate for proprotein convertases such as furin. To determine the role of furin and other proprotein convertases, we expressed proBACE in a furin‐deficient mutant Chinese hamster ovary (CHO‐K1) line, RPE.40. ProBACE signal was higher in RPE.40 than in the CHO‐K1 parent, which confirmed that furin plays a role in propeptide removal. However, two independent approaches showed that proBACE is cleaved to mature BACE in RPE.40: proBACE was rapidly turned over in RPE.40 although total BACE was stable, and decanoyl‐RVKR‐chloromethylketone, an inhibitor of the proprotein convertase family, substantially increased proBACE levels in both RPE40 and CHO‐K1. Transient transfection shows that furin, PACE4, PC5/6, and PC7 mediate BACE cleavage in vivo , at least when overexpressed. RPE.40 is proficient in BACE activity despite its furin deficiency. Therefore, our finding that proBACE is cleaved in this mutant leaves open the possibility that maturation is an important regulatory step and a therapeutic target.

ABSTRACT The abnormal accumulation of the amyloid β protein (Aβ) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Aβ accumulation may therefore be useful therapeutically. In cell‐based screens we detected a significant reduction in Aβ concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Aβ accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non‐overlapping 40%–50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Aβ in the brain of treated animals indicates that both Aβ40 and the longer, more amyloidogenic form of the peptide, Aβ42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Aβ concentration in vitro can reduce Aβ accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Aβ accumulation.

ABSTRACT The amyloid β‐protein (Aβ) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid β‐protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events: an initial β‐secretase cleavage at the amino terminus of the Aβ sequence followed by γ ‐secretase cleavage at the carboxyl terminus of Aβ. We describe the development of a robust in vitro assay for γ ‐secretase cleavage by showing de novo Aβ production in vitro and establish that this assay monitors authentic gamma‐secretase activity by documenting the production of a cognate γ ‐CTF, confirming the size of the Aβ produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter γ‐ secretase activity in living cells. Using this assay, we demonstrate that the γ‐secretase activity 1 ) is tightly associated with the membrane, 2 ) can be solubilized, 3 ) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4 ) ascertain that activities of the γ ‐40 and γ ‐42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3− > Cl− > HCO3− > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo . Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.