We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thyrnocytes. o 1993 Wiley-Liss, Inc. Key terms: Apoptosis, flow cytometry, thymocytesEukaryotic cells are thought to die either by necrosis or apoptosis. Loss of integrity of the cell membrane is an early event in necrosis while in apoptosis this is preceded morphologically by condensation of nuclear chromatin, compaction of cytoplasmic organelles, and changes at the cell surface (1,421. Biochemically, the most striking change in apoptosis is the digestion of nuclear DNA into oligonucleosomal length fragments (41). Apoptosis can be induced in immature thymocytes in vitro by a variety of agents including glucocorticoids, such as dexamethasone (41,431 and compounds reactive with DNA topoisomerase 11, for example, etoposide (37).In some types of cell, apoptotic and normal cells can be distinguished by a brief incubation with the bisbenzimidazole dye, Hoechst 33342; the apoptotic cells fluoresce more brightly on excitation of the dye-DNA complex by uv radiation (9,23). In the same assay, cells which have lost the integrity of their plasma membrane can be separately quantified by their failure to exclude another DNA binding dye, propidium iodide (PI). We have adapted this method to quantify viable apoptotic rat thymocytes (30); the method has also assisted us in the recognition, isolation, and characterisation of a transitional preapoptotic population (4).Hoechst 33342 is one of a family of bis-benzimadazoles which stain DNA specifically, binding preferentially to repetitive AT sequences in the minor groove of the helix (2,191. The fluorescence of the dye in this environment is an order of magnitude greater than its fluorescence in aqueous solution. At high concentrations of dye, there is a second mode of binding which leads to fluorescence quenching (2) and a red shift of the fluorescence spectrum (28,29,39). Hoechst 33342 is taken up by viable cells although the rate of uptake into the nucleus strongly depends on the type of cell studied and this property has been used to distinguish different types of lymphoid cell (16,391. The dye is actively pumped out of cells by an energy dependent pump which is probably the p-glycoprotein pump responsible for multiple drug...

The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure. Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance. A cluster of overexpressed genes related to DOX resistance was observed. Included in this cluster was ABCB1 the Pglycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target. Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity. In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identi®ed which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway. Analysis of genomic ampli®cations and deletions revealed speci®c genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK. The ®ndings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention. Oncogene (2001) 20, 1300 ± 1306.

Summary The potent kinase inhibitor staurosporine and its protein kinase C (PKC)-selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by Pglycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF-7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nm, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nm were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P-gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp-mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development.

BALB/c Fech m1Pas mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism Heme is vital to the transport and utilization of oxygen in oxidative and signaling pathways. A large proportion of heme usage in the liver is accounted for by cytochrome P450 isoforms, many of which are associated with drug metabolism. In the final step of heme synthesis ferrous iron is inserted into the precursor porphyrin protoporphyrin IX by the mitochondrial enzyme ferrochelatase. The structure and regulation of ferrochelatase have been extensively studied.

It has been suggested that the thyroid itself may contribute to the inflammatory process observed in autoimmune thyroiditis by releasing the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), but studies of cytokine gene expression in thyrocytes have been limited and conflicting. A semi-quantitative reverse transcription-PCR technique has been used to investigate the expression of IL-1 alpha, IL-6 and IL-8 mRNA in the human thyroid cell line HTori3 and in cultures of primary human thyroid follicular cells (TFCs). Cytokine mRNA levels were examined over a 24-h period, and the modulatory effects of exogenous IL-1 alpha, interferon-gamma (IFN-gamma) and TSH investigated. Basal expression of IL-1 alpha, IL-6 and IL-8 mRNA was detected in HTori3 and primary TFC cultures. Stimulation with IL-1 (10 U/ml) for 12 h produced an increase in the level of IL-1 alpha mRNA in both primary TFC and HTori3 cultures. IL-6 and IL-8 mRNA levels were increased by the addition of IL-1 in both cell types, and this effect was detected throughout the 24-h time-course. IFN-gamma (100 U/ml) had no significant effect on cytokine gene expression. A higher concentration of IFN-gamma (500 U/ml) had no significant effect on the expression of IL-1 alpha or IL-8 but produced an increase in the level of IL-6 mRNA in primary cultures and in HTori3 cells. Addition of TSH (1 mU/ml) produced an increase in the level of IL-1 alpha mRNA in primary TFC and HTori3 cells, at 12 and 24 h. TSH had no significant effect on the expression of IL-6 or IL-8 mRNA. These results demonstrate that human TFCs constitutively express IL-1 alpha, IL-6 and IL-8 mRNA and that this expression can be modulated by IL-1, IFN-gamma and TSH.

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GLYCOGEN SYNTHESIS IN RAT DIAPHRAGM 6097. Glycogenolysis in diaphragm was favoured by potassium but unaffected by insulin. The order of magnitude of glycogenolysis was the same in 'normal' and 'diabetic' diaphragm.8. Metabolic changes of tissues in vitro may become manifest only in a correspondingly changed medium and may be undetectable in an indifferent salt medium. This investigation was aided by a grant of the Dazian Foundation for Medical Research and by the Sam and H. Ansel Moskowitz Fund for research in diabetes.

Polymorphisms of genes linked to iron metabolism may account for individual variability in hemochromatosis and iron status connected with liver and cardiovascular diseases, cancers, toxicity, and infection. Mouse strains exhibit marked differences in levels of non-heme iron, with C57BL/6J and SWR showing low and high levels, respectively. The genetic basis for this variability was examined using quantitative trait loci (QTL) analysis together with expression profiling and chromosomal positions of known iron-related genes. Non-heme iron levels in liver and spleen of C57BL/6J ؋ SWR F 2 mice were poorly correlated, indicating independent regulation. Highly significant (P < .01) polymorphic loci were found on chromosomes 2 and 16 for liver and on chromosomes 8 and 9 for spleen. With sex as a covariate, additional significant or suggestive (P < 0.1) QTL were detected on chromosomes 7, 8, 11, and 19 for liver and on chromosome 2 for spleen. A gene array showed no clear association between most loci and differential iron-related gene expression. The gene for transferrin and a transferrin-like gene map close to the QTL on chromosome 9. Transferrin saturation was significantly lower in C57BL/6J mice than in SWR mice, but there was no significant difference in the serum level of transferrin, hepatic expression, or functional change in cDNA sequence. ␤2-Microglobulin, which, unlike other loci, was associated with C57BL/6J alleles, is a candidate for the chromosome 2 QTL for higher iron. In conclusion, the findings show the location of polymorphic genes that determine basal iron status in wild-type mice. Human equivalents may be pertinent in predisposition to hepatic and other disorders. (HEPATOLOGY 2006;44:174-185.)