Intracellular labile iron pools as direct targets of iron chelators: a fluorescence study of chelator action in living cells

The primary targets of iron chelators used for treating transfusional iron overload are prevention of iron ingress into tissues and its intracellular scavenging. The present study was aimed at elucidating the capacity of clinically important iron chelators such as deferiprone (DFP), desferrioxamine, and ICL670 to (a) gain direct access to intracellular iron pools of key cells of iron accumulation (macrophages, hepatocytes, and cardiomyocyte cell lines); (b) chelate the labile iron present in discrete cell compartments/organelles; and (c) prevent labile iron involvement in the generation of reactive oxidant species. Chelation of cytosolic and organellar cell iron was visualized dynamically and quantitatively in living cells by fluorescence microscopic imaging of fluorescent metallosensors (used as iron-quenched complexes of calceins) targeted to either cytosol, endosome-lysosomes, or mitochondria. The rate and extent of fluorescence recovery provided an in situ measure of the accessibility of chelators to particular cell sites/organelles. Complementary, fluorogenic redox probes associated with cell compartments enabled identification of chelator-sensitive, localized reactive oxidant production. Our studies indicate that chelation by desferrioxamine is slow and is enhanced in cells with relatively high endocytic activities, while ICL670 and DFP readily enter most cells and efficiently reach the major intracellular sites of iron accumulation.     

BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study

There is a clear association between germ-line BRCA1 mutations and inherited ovarian cancer; however, the association between BRCA1 mutations and sporadic ovarian cancer remains ambiguous. The frequency of BRCA1 promoter hypermethylation as an epigenetic means of BRCA1 inactivation was determined for a large, population-based cohort of ovarian cancer patients. BRCA1 promoter hypermethylation was determined by methylation-specific restriction digestion of tumor DNA, followed by Southern blot analysis and confirmed by methylation-specific PCR. BRCA1 promoter hypermethylation was observed in 12 of 98 ovarian tumors. BRCA1 methylation status of the primary tumor was conserved in six recurrent tumors after interim chemotherapy. None of the 12 tumors with BRCA1 promoter hypermethylation demonstrated BRCA1 protein expression by immunohistochemistry. BRCA1 methylation was only seen in ovarian cancer patients without a family history suggestive of a breast/ ovarian cancer syndrome. Therefore, the 12 BRCA1 methylated tumors represented 15% (12 of 81) of the sporadic cancers analyzed in this study. Although the clinical significance of BRCA1 promoter hypermethylation is yet to be determined, promoter hypermethylation may be an alternative to mutation in causing the inactivation of the BRCA1 tumor suppressor gene in sporadic ovarian cancer.     

Antidepressants induce cellular insulin resistance by activation of IRS-1 kinases

Certain selective serotonin reuptake inhibitors (SSRIs) induce the clinical and biochemical manifestations of a metabolic syndrome by as yet unknown mechanism. Here we demonstrate that incubation (1 h) of rat hepatoma Fao cells with the SSRIs paroxetine and sertraline, but not with the atypical antipsychotic drug olanzapine, inhibited the insulin-stimulated Tyr phosphorylation of the insulin receptor substrate-1 (IRS-1) with half-maximal effects at approximately 10 microM. This inhibition correlated with a rapid phosphorylation and activation of a number of Ser/Thr IRS-1 kinases including JNK, S6K1, ERK and p38 MAPK, but not PKB (Akt). JNK appears as a key player activated by SSRIs because specific JNK inhibitors partially eliminated the effects of these drugs. The SSRIs induced the phosphorylation of IRS-1 on S307 and S408, which inhibits IRS-1 function and insulin signaling. These results implicate selected SSRIs as inhibitors of insulin signaling and as potential inducers of cellular insulin resistance.     

Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct

Developing advanced technologies for encouraging the ex vivo assembly of functional hepatic tissue for implantation into the human body or for in vitro drug testing is one of the challenging tasks facing tissue engineers. In the present study, we utilized a perfusion bioreactor system equipped with a novel flow-distributing mesh for online cell seeding into macroporous alginate scaffolds and cultivation of multiple constructs of the C3A human hepatocyte cell line. Optimization of the medium flow rate (100 mL/min) and perfusion duration (12 h) yielded cell constructs with high cell seeding efficiency (98% of the input cells) and cell distribution throughout the entire scaffold. Further, we show that interstitial medium flow enabled uniform cell delivery into 35 constructs lined across the bioreactor cross section. Perfusion-cultivated cell constructs revealed much greater rates of cell proliferation, albumin-specific secretion, and gene expression of the phase I enzyme, CYP3A4, and phase II enzyme, UGT2B7, than did static-cultivated constructs. Most impressive was the 50-fold increase in CYP3A4 expression of the perfused cell constructs as compared to the level in static-cultivated cell constructs. We thus believe that the hepatic tissue constructs developed herein may be used in drug discovery programs for elucidating drug metabolism and toxicity profiles and for treating failing livers.     

Expression of the human α-1-antitrypsin gene in heterologous mammalian cells

A genetic-engineering construction is developed containing the full-size cDNA of human α-1-antitrypsin, controlled by the promotor and enhancer elements from cytomegalovirus. It is shown that, after transfection with this recombinant DNA, it is properly expressed in heterologous animal cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, vol. 117, N ^o 2, pp. 166–167, Feburary, 1993 Presented by A. N. Klimov, Member of the Russian Academy of Medical Sciences