Defective acidification in human breast tumor cells and implications for chemotherapy

Multidrug resistance (MDR) is a significant problem in the treatment of cancer. Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles. Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles. This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy. These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.     

Financing and payment reform for primary health care and substance abuse treatment

One of the most controversial areas for health care reform concerns the treatment of alcohol and other drug problems, which account for some of the most rapidly rising costs in the health care sector. There is arguably no other set of conditions that show such variation in accessibility to treatment on the basis of insurance status, present the same degree of difficulty in providing comprehensive care, or challenge as many public and professional assumptions about behavioral, social and economic determinants. The purpose of this article is to discuss some of the financing and coverage barriers to comprehensive treatment for alcohol and other drug abuse; to discuss some innovative mechanisms for providing and financing comprehensive services; and to suggest some directions for public policy to support the development of new practice models that emphasize cost-effectiveness and efficiency of care.     

Control of smooth muscle cell proliferation by psoralen photochemotherapy

PURPOSE: Restenosis after balloon angioplasty or the intimal hyperplasia occurring at distal anastomoses of bypass grafts severely limits the long-term therapy for peripheral vascular disease. The aim of this study was to evaluate the application of psoralen photochemotherapy with ultraviolet A (UVA)-activated 8-methoxypsoralen (8-MOP) to suppress smooth muscle cell (SMC) proliferation in vitro by the formation of 8-MOP-DNA monoadducts and interstrand cross-links to inhibit DNA synthesis. METHODS: Bovine aorta SMC (2 x 10(4)/cm2) were treated with 8-MOP (0 to 1000 ng/ml) for 30 minutes, followed by UVA (2 joule/cm2) to determine the dose of 8-MOP and UVA that inhibits SMC proliferation. RESULTS: The results show that 8-MOP in the range 30 to 1000 ng/ml in combination with 2 joule/cm2 UVA inhibited SMC proliferation by 40% to 60% 3 days after treatment. In time course studies the growth of SMC treated with 100 ng/ml 8-MOP and 2 joule/cm2 UVA were monitored over 5 days, and this regimen was found to be cytostatic. SMC viability was confirmed by trypan blue exclusion. CONCLUSIONS: Our studies suggest that 8-MOP/UVA photochemotherapy may represent a novel approach to the control of localized SMC proliferation.     

Impaired glucose tolerance, type 2 diabetes, and carotid wall thickness: the Insulin Resistance Atherosclerosis Study

OBJECTIVE: To assess whether people with impaired glucose tolerance (IGT) exhibit an increased risk of atherosclerosis as measured by the thickness of the carotid artery. RESEARCH DESIGN AND METHODS: We examined the relationship between glucose tolerance status and subclinical atherosclerosis in the Insulin Resistance Atherosclerosis Study (IRAS). The IRAS is an epidemiological study of 1,625 Hispanic, African-American, and white men and women, with approximately equal numbers of subjects with normal glucose tolerance (NGT), IGT, and type 2 diabetes as assessed by an oral glucose tolerance test. Half of those with diabetes were previously unaware of their condition and were defined as having new diabetes. Persons using insulin were excluded. The intima-media thickness (IMT) of the common carotid artery (CCA) and internal carotid artery (ICA) was measured as an index of subclinical atherosclerosis using B-mode ultrasonography. RESULTS: Adjusted for demographics and smoking, CCA-IMT increased most notably at the level of established diabetes (802, 822, 831, and 896 microm for NGT, IGT, new diabetes, and established diabetes, respectively). Adjustment for coronary heart disease (CHD) risk factors, which tended to worsen across glucose tolerance category, further minimized the slightly graded relationship. The relationship with the ICA-IMT was steeper and again suggested that the increased wall thickness is associated with diabetes, not with IGT. The relationship between glucose tolerance category and IMT was similar in men and women. CONCLUSIONS: We observed considerably greater IMT among persons with established diabetes but no significant increase in persons with IGT. These data suggest that the increased risk of CHD observed in persons with diabetes may largely develop after the onset of overt diabetes.     

The -514 polymorphism in the hepatic lipase gene (LIPC) does not influence androgen-mediated stimulation of hepatic lipase activity

The -514T allele of hepatic lipase is associated with increased high density lipoprotein-cholesterol levels in men, but not in women. This observation suggests that the -514C to T polymorphism may diminish the response of hepatic lipase to androgens. To test this hypothesis, five -514T and five -514C homozygous men were treated with the anabolic steroid stanozolol for 6 days. The mean increase in hepatic lipase activity was similar in the two groups (45+/-10 vs. 51+/-10 mmol x hr(-1) x l(-1), P = 0.5). To evaluate the association between the -514 polymorphism and hepatic lipase activity at different physiological androgen concentrations, hepatic lipase genotypes and activities were measured in 44 men and 40 premenopausal women. The effect of the -514T allele on hepatic lipase activity was significant and quantitatively similar in both sexes. These data indicate that the -514 polymorphism does not influence the response of hepatic lipase activity to androgens, and that the effects of this polymorphism on hepatic lipase activity are independent of androgen action.     

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.     

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.     

Mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection

IFN-gamma is critical in the immune response to mycobacterial infections, and deficits in IFN-gamma production and response have been associated with disseminated nontuberculous mycobacterial infections. Mutations in the IFN-gamma receptor ligand-binding chain (IFNgammaR1) have been shown to confer susceptibility to severe infection with nontuberculous mycobacteria. However, mutations in the IFN-gamma receptor signal-transducing chain (IFNgammaR2) have not been described. We describe a child with disseminated Mycobacterium fortuitum and M. avium complex infections and absent IFN-gamma signaling due to a mutation in the extracellular domain of IFNgammaR2. In vitro cytokine production by patient PBMCs showed 75% less PHA-induced IFN-gamma production than in normal cells, while patient PHA-induced TNF-alpha production was normal. The normal augmentation of TNF-alpha production when IFN-gamma was added to endotoxin was absent from patient cells. Expression of IFNgammaR1 was normal, but there was no phosphorylation of Stat1 in response to IFN-gamma stimulation. DNA sequence analysis of the gene for IFNgammaR2 showed a homozygous dinucleotide deletion at nucleotides 278 and 279, resulting in a premature stop codon in the protein extracellular domain. This novel gene defect associated with disseminated nontuberculous mycobacterial infection emphasizes the critical role that IFN-gamma plays in host defense against mycobacteria.