Enhancement of the activity of phenoxodiol by cisplatin in prostate cancer cells.

Phenoxodiol is a novel isoflav-3-ene, currently undergoing clinical trials, that has a broad in vitro activity against a number of human cancer cell lines. Phenoxodiol alone inhibited DU145 and PC3 in a dose- and time-dependent manner with IC(50) values of 8+/-1 and 38+/-9 microM, respectively. The combination of phenoxodiol and cisplatin was synergistic in DU145, and additive in PC3, as assessed by the Chou-Talalay method. Carboplatin was also synergistic in combination with phenoxodiol in DU145 cells. The activity of the phenoxodiol and cisplatin combination was confirmed in vivo using a DU145 xenograft model in nude mice. Pharmacokinetic data from these mice suggest that the mechanism of synergy may occur through a pharmacodynamic mechanism. An intracellular cisplatin accumulation assay showed a 35% (P<0.05) increase in the uptake of cisplatin when it was combined in a ratio of 1 microM:5 microM phenoxodiol, resulting in a 300% (P<0.05) increase in DNA adducts. Taken together, our results suggest that phenoxodiol has interesting properties that make combination therapy with cisplatin or carboplatin appealing.

Alterations of platelet aggregation kinetics with ultraviolet laser emission: the "stunned platelet" phenomenon.

Platelets, a major constituent of thrombus, play a crucial role in the pathogenesis of acute ischemic coronary syndromes. The effect of ultraviolet laser emission on platelets within thrombi is unknown. The effects of increasing levels of laser energy on platelets in whole blood were investigated. Blood samples were obtained by aseptic venipuncture and anticoagulated with 3.8% sodium citrate. Samples were exposed to increased levels (0, 30, 45, 60 mJ/mm2; 25 Hz) of ultraviolet excimer laser fluence (308 nm wave-length) and then tested for ADP and collagen induced platelet aggregation, platelet concentration, and for platelet contractile force (PCF) development. Scanning electron microscopy was used to detect laser induced morphologic changes of platelets and by flow cytometric analysis to detect changes in expression of platelet surface antigens p-selectin (CD 62) and glycoprotein IIb/IIIa (CD 43). Exposure to excimer laser energy produced dose dependent suppression of platelet aggregation and force development ("stunned platelets"). ADP aggregation decreased from 8.0+/-1.1 Ohms (mean+/-SEM) to 3.7+/-0.8 Ohms (p<0.001) to 2.7+/-0.6 Ohms (p <0.001) and to 1.8+/-0.5 Ohms (p <0.001) as the laser energy increased from 0 to 30 to 45 to 60 mJ/mm2, respectively. Collagen induced aggregation decreased from 21.4+/-1.4 Ohms to 15.7+/-1.2 Ohms (p <0.001) to 11.7+/-1.1 Ohms (p <0.001) and to 9.9+/-1.0 Ohms (p <0.001), in response to the same incremental range of laser energy. Platelet contractile forces declined from 34,500+/-3700 to 27.800+/-2700 dynes as laser energy increased from 0 to 60 mJ/mm2 (p <0.03). Platelet concentration did not change with increasing laser energy. The expression of platelet surface antigen p-selectin (CD 62) remained stable through increasing levels of laser energy exposures while the percentage of CD 43 positive platelets significantly increased with exposure to laser energy, yet the level of expression did not exceed 0.5% of cells. Thus, aggregation kinetics are altered in platelets exposed to ultraviolet laser energy as manifested by decreased platelet aggregation and reduction in platelet force development capability. The response is dose dependent and most pronounced at higher energy levels such as 60 mJ/mm2.

Effect of long-term estrogen deprivation on apoptotic responses of breast cancer cells to 17beta-estradiol.

BACKGROUND: High doses of estrogen can promote tumor regression in postmenopausal women with hormone-dependent breast cancer, but the mechanism is unknown. We investigated the molecular basis of this process by using LTED cells, which were derived by growing MCF-7 breast cancer cells under long-term (6-24 months) estrogen-deprived conditions. METHODS: We treated LTED and MCF-7 cells with various concentrations of 17beta-estradiol (estradiol) and assayed their growth by counting the cells and measured apoptosis by annexin V staining and DNA fragmentation. Using western blot analysis, we also examined the expression of the apoptosis-inducing system of the Fas death receptor protein and its ligand, FasL, in these cells. To assess the involvement of Fas and FasL in the induction of apoptosis in LTED cells, we used activating anti-Fas antibodies and the universal caspase inhibitor Z-VAD. Finally, we examined the expression of Fas protein in E8CASS and BSK3 cells, two other cell lines derived by depriving MCF-7 cells of estrogen long term, and the responses of these cells to high-dose estradiol. All statistical tests were two-sided. RESULTS: High concentrations of estradiol (>or=0.1 nM) resulted in a statistically significant, 60% reduction in the growth of LTED cells (P< .001) and in a sevenfold increase in apoptosis (P< .001) as compared with levels in vehicle-treated cells. Both LTED and MCF-7 cells expressed FasL, but only LTED cells expressed Fas. Treatment of LTED cells with 0.1 nM estradiol increased the expression of FasL. Activating anti-Fas antibodies increased apoptosis of LTED cells, which was further stimulated by estradiol. Z-VAD blocked estradiol-induced apoptosis. E8CASS cells, which express Fas protein, but not BSK3 cells, which do not, also responded to 0.1 nM estradiol by increasing apoptosis. CONCLUSION: Tumor regression induced by high-dose estrogen therapy in postmenopausal woman may result from estrogen activation of Fas-mediated apoptosis.

Commentary: advances in the laboratory diagnosis of celiac disease.

Anti-endomysial autoantibodies are very useful in the diagnosis and monitoring of celiac disease (gluten-sensitive enteropathy) due to their high sensitivity and specificity for that disorder. The recent discovery that the autoantigen responsible for the endomysial pattern is tissue transglutaminase (tTG) has led to the commercial development of automated enzyme immunoassays for quantitation of that autoantibody. These assays are standardized to provide highly accurate and comparable testing between laboratories for anti-tTG autoantibodies. Celiac disease is a common genetic disease in populations of Europe and the United States. It has a spectrum of expression ranging from silent or mild to severe, with resulting malabsorption that produces multiple-organ system effects due to malnutrition. Many cases miss the diagnosis because the symptoms are not classic or the clinical syndrome is not severe. Because the treatment of celiac disease (avoidance of wheat products) is so effective and inexpensive and because celiac disease is so common in selective populations, a highly reliable test for its detection such as anti-tTG should find wide application in clinical practice.

Contrasting effects of acute and chronic dietary exposure to 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) on xenobiotic metabolising enzymes in the male Fischer 344 rat: implications for chemoprevention studies.

BACKGROUND: 2-Amino-3-methyl-imidazo[4,5-f]quinoline (IQ) is a mutagen produced in cooked food. It is commonly present in the human diet and often used as a (pro)carcinogen for chemoprevention studies. Many foodstuffs act as chemopreventers by altering xenobiotic metabolising enzyme expression in favour of detoxication over bioactivation pathways. However, IQ itself can also affect enzyme expression, which may be a confounding factor in chemoprevention studies. AIM OF STUDY: Chronic low dose IQ exposure is intuitively closest to the human dietary situation. The aim was to investigate the effects of chronic dietary exposure to IQ on the expression of enzymes involved in the bioactivation and detoxification of xenobiotics and to compare this with acute exposure, often used in chemoprevention studies. METHODS: Male Fischer rats received IQ (300 ppm) in the diet (AIN-76) for 52 weeks or were given IQ (20 mg.kg-1) orally for 3 days. Animals were killed, livers removed and subcellular fractions prepared. A range of enzymes was selected to allow investigation of several cellular mechanisms. Enzyme expression and activity were determined by Western blotting and the use of selective probe substrates as appropriate. RESULTS: Chronic exposure to IQ led to an increase in phase II detoxifying enzymes. Both the activity and expression of glutathione S-transferase (GST-A1/2) were increased, as were NADPH: Quinone oxidoreductase (NQO), UDP-glucuronosyl transferase (UGT) and beta-glucuronidase activities. There were no statistically significant changes in the potential for bioactivation by three cytochrome P450s. In contrast, acute IQ exposure significantly increased the expression and activity of some cytochrome P450 (CYP1A1 and CYP1A2), UGT and beta-glucuronidase, but significantly decreased glutathione S-transferase expression and activity. There was a non-significant decrease in NQO but no change in CYP3A2 and CYP2E1 activities. CONCLUSIONS: The changes after acute exposure suggest an interaction through the Ah receptor and xenobiotic response element, modified by the glucocorticoid response element. In contrast, the pattern of effects after chronic exposure suggests activation of the antioxidant response element (ARE). Although the acute model is more practically convenient for short-term chemoprevention screening, the data suggest that an entirely new mechanism is being invoked that completely masks effects of the ARE that occur during chronic exposure. There is a danger that chemopreventive strategies developed using acute models may be misleading, since the mechanism is unlikely to occur during human dietary exposures.

Blood sample volumes: emerging trends in clinical practice and laboratory medicine.

Blood loss because of phlebotomy for diagnostic laboratory tests is a well-recognized risk to neonates, particularly low-birthweight infants. In contrast, the risk of anemia from blood drawing in adults is relatively poorly studied. A few clinical studies have demonstrated the magnitude of this issue for critical care patients; most adult patients can easily tolerate the loss of blood volumes typically used for laboratory tests. Recommendations for promoting blood conservation in adults who frequently are phlebotomized include using smaller collection tubes, but more importantly organizing blood draws to eliminate duplicate and other unnecessary test requests and consolidating multiple collections into as few as possible by scheduling them. Emerging trends in medicine that will bear on the practice of blood conservation are "bloodless surgery," standardization of collection tubes for laboratory automation systems, and smart laboratory information systems that provide instant feedback to ordering physicians.

A different function for a critical tryptophan in c-Raf and Hck.

The similarity of the catalytic domains of Raf and Src family members suggests that functions of homologous residues may be similar in both kinase families. A tryptophan residue, W260, in the WEI region of the Src family kinase Hck has an important role in regulating ATP binding. We tested the hypothesis that the tryptophan, W342, in the WEI region of c-Raf may have a similar role to the W260 of Hck. Mutation of W260 to A in Hck activates kinase activity, but we found that mutation of W342 to A in c-Raf inactivates the kinase activity. Mutating W342 to aspartate (D), lysine (K) or histidine (H) also inactivated c-Raf whether assayed as a purified immunoprecipitate or when recruited to the plasma membrane. A constitutively active c-Raf can be generated by mutating two regulatory tyrosines to aspartate. When placed into this active c-Raf mutant, mutation of W342 to D, K or H enabled phosphorylation and activation of the c-Raf substrate MEK at the plasma membrane but not in an immunoprecipitation assay. We conclude that (1) Tryptophan has a different role in the WEI regions of c-Raf and Hck, (2) W342 is not directly involved in MEK binding as both positive and negative residues at 342 are permissive for MEK activation at the membrane in a constitutively active c-Raf mutant, (3) Factors at the membrane are capable of potentiating activation of c-Raf containing mutated W342 in a hyperactivated c-Raf, but not in a wild type c-Raf and (4) There is a stringent structural requirement for W at residue 342 in c-Raf.

Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.

Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.

Perspective on the clinical laboratory: new uses for informatics.

Enhanced capabilities of modern information systems will have a major impact on the way that clinical laboratories generate diagnostic information and transmit that information to their physician clients. This article provides a perspective on how some of the most imminent changes in informatics are likely to alter laboratory practices and to create new roles for the clinical laboratory through the ability to manage vast quantities of information. The areas covered include utilization of laboratory services, process control in the laboratory, interpretation of test results, and laboratory economics.

Robotics, automation, and the new role of process control.

The natural progression of automation in the clinical laboratory next will lead to robotic devices to perform many of the manual tasks still remaining. To date, most efforts of laboratory automation have been directed at the analytic phase. New targets for automation will be at the preanalytic and postanalytic phases where many of the bottlenecks in specimen flow now occur in highly repetitive manual tasks. Laboratory professionals will have a unique opportunity to incorporate new concepts of robotics in their facilities to improve error rates and to use massive laboratory databases to improve medical and public health services.

14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo.

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

Evaluation of the Sysmex UF-100 automated urinalysis analyzer.

Urinalysis is a high-volume procedure that currently requires significant labor to examine microscopic sediment. We evaluated the Sysmex UF-100 automated urinalysis analyzer for performing this task. Instrument accuracy was assessed by comparing continuous counts of microscopic elements from the UF-100 with ranges of cells (per low-power field or high-power field) from manual microscopy performed on centrifuged urines. Counts showed good agreement between methods (gamma statistic: 0.880-0.970) for all microscopic elements in 252 urine samples. Within-run imprecision of cell counts expressed as CV (mean cell count/microL) was for erythrocytes (RBC) 31% (5), 18% (50), 2.4% (800); for leukocytes (WBC) 14% (10), 11% (100), 8.5% (400); for squamous epithelial cells (SEC) 18% (5), 12% (30), 7.0% (100); for casts 45% (1), 17% (4); for bacteria 2-12% (entire range of 40-2500). Between-run imprecision on quality-control cell suspensions expressed as CV (mean cell count/microL) was for RBC 6.1% (50), 2.7% (256); for WBC 26.9% (54), 4.9% (228). Cells counted on dilution were 99.1% of expected for RBC, 102.0% for WBC, and 121.8% for bacteria. Carryover was <0.04% for RBC, <0.03% for WBC, <0.14% for SEC, <0.29% for bacteria. We conclude that the UF-100 can automatically perform reliable quantitative microscopic urinalysis in batches without operator interaction.

Cost-effectiveness of cardiac troponin I in a systematic chest pain evaluation protocol: use of cardiac troponin I lowers length of stay for low-risk cardiac patients.

We evaluated several measures of clinical and fiscal interest to assess the effect of adding an automated cardiac troponin I (c-TnI) assay to our current cardiac panel, which consists of creatine kinase MB (CK-MB), myoglobin, total CK activity, and a calculated CK-MB relative index. Samples were collected on admission and at 3, 6, and 8 hours after admission as part of our diagnostic protocol. Our study was designed to collect data on a control group of patients, implement a change (i.e., c-TnI testing), and then measure the effect of the change on a test population having otherwise equivalent diagnostic and therapeutic pathways. We assessed differences in patient hospital and cardiac care unit length of stay (LOS), time to cardiac catheterization, and hospital and laboratory charges and costs. We found that adding c-TnI to our testing regimen decreased LOS for the large test population. Within this large test population, patients classified as low risk for acute myocardial infarction experienced statistically and clinically significant shorter LOS and lower total and variable hospital costs; for patients with unstable angina, there was an increase (though not statistically significant) in laboratory costs.

Molecular basis of genetic disease and molecular methods.

Nucleic acid testing is possible because of the physical and biomechanical properties of DNA and RNA such as hybridization, which also form the basis for the physiologic role of nucleic acids. The entire framework of genetic analysis involves the nucleotide sequence of genes, intragenic structure, supragene organization, and mapping of genes onto chromosomes. Genetic diseases are the consequence of mutations affecting one to many nucleotides. Such genetic alterations are detectable by several special assays for examining nucleic acids that are now common in genetic laboratories.

Ethical issues in screening and testing for genetic diseases.

New analytic techniques coupled with detailed knowledge of the entire human genome soon will make possible testing of all genes in every person. Concepts of disease will change, and predisease states will be identified for genetic therapies. Patients, physicians, and laboratory personnel will be confronted with ethical and moral issues in the performance of genetic testing. The primary ethical issues will focus on who has the right to request or compel genetic testing, who has access to confidential information, and what medical or social actions may be predicated legally on genetic information.

Alterations in erythrocyte band 3 organization induced by the photosensitizer, hematoporphyrin derivative.

Photosensitization of erythrocytes in the presence of hematoporphyrin derivative causes cross-linking of membrane proteins. This cross-linking is associated with partial lysis of the cells and an increased susceptibility to heat-induced membrane fragmentation. The effect of photosensitization on the organization of erythrocyte band 3 was monitored using the technique of time-resolved phosphorescence anisotropy. Band 3 rotational diffusion was somewhat restricted upon photooxidation, indicating aggregation of this major integral membrane protein.

Laboratory diagnosis of human hepatitis viruses.

Conventional serologic methods of antigen or antibody detection are now widely applied for diagnosis of hepatitis viruses A, B, C, and D. Nucleic acid quantitation has become very useful for monitoring response to antiviral therapy in cases of hepatitis B and C. Special confirmatory testing of HCV serologies can be quite specific, but overall serologies for HCV lack sensitivity for early diagnosis. Thus HCV RNA detection may ultimately be the preferred method for HCV diagnosis and for screening blood donors. Unfortunately, HEV diagnosis may rest on the efforts of research laboratories for electron microscopy, Western blot, or nucleic acid detection.

Anti-glycosphingolipid autoantibodies in rheumatologic disorders.

Antibodies directed against ganglioside GM1 or sulfatides are frequently associated with motor or sensorimotor neuropathies. To establish the prevalence of such anti-glycosphingolipid autoantibodies in autoimmune disorders and to determine whether they contribute to neurologic symptoms in those individuals, we measured these antibodies by enzyme-linked immunosorbent assay (ELISA) in serum samples from rheumatologic patients with and without peripheral neuropathies (PN). We tested 21 patients with systemic lupus erythematosus (9 with PN), 26 with Sjögren's syndrome (12 with PN), 34 with scleroderma (28 with PN), and 14 with rheumatoid arthritis (4 with PN). Samples from 32 normal individuals were also tested. Patients with systemic lupus erythematosus and rheumatoid arthritis had elevated concentrations of GM1 antibodies and scleroderma patients had lower levels of sulfatide antibodies compared to healthy individuals. The presence of ganglioside or sulfatide antibodies did not correlate with the development of peripheral neuropathy in these patients. These findings suggest that relatively low-titer glycosphingolipid antibodies may arise as part of a nonspecific polyclonal gammopathy in rheumatologic disorders but generally without clinical manifestation.