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.

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.

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.

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.

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.

Structural organisation of band 3 in Melanesian ovalocytes.

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normocytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.

Proteolytic digestion of band 3 at an external site alters the erythrocyte membrane organisation and may facilitate malarial invasion.

Invasion of human erythrocytes by Plasmodium falciparum is inhibited by chymostatin. This suggests that digestion of erythrocyte surface proteins by a protease with chymotrypsin-like activity may be involved in the invasion process. We find that treatment of intact erythrocytes with chymotrypsin cleaves the integral membrane protein, band 3, generating a major fragment with an apparent molecular weight of 58 kDa. We have used measurements of the rotational mobility of band 3, labelled with the phosphorescence probe, eosin-5-maleimide, as a monitor of the changes in the molecular organisation of the erythrocyte membrane which accompany band 3 cleavage. We report that the chymotrypsin treatment increases the rotational freedom of band 3, possibly due to conformational changes which disrupt its interaction with the underlying peripheral membrane proteins. We also show that chymotrypsin-treated erythrocytes undergo extensive endocytosis upon incorporation of exogenous fluorescently labelled phospholipid. We suggest that during the invasion process, digestion of band 3 by a chymotrypsin-like protease may induce a localised disruption of the erythrocyte membrane. This destabilised region of membrane may represent the site for the insertion of parasite-derived phospholipid, thus allowing the formation of the parasitophorous vacuole membrane.

Evidence of a cyclosporine-binding protein in human erythrocytes.

To investigate the phenomenon of different erythrocyte saturation capacities for cyclosporine (CsA) in the blood of different individuals, hemolysates of washed red cells were examined for the presence of a CsA-binding protein. Using gel filtration column chromatography of hemolysates from patients receiving CsA orally, the majority of erythrocyte-associated CsA eluted as a single peak with Mr 15,000-17,000, distinct from hemoglobin and carbonic anhydrase. [3H]CsA added to a hemolysate in vitro eluted similarly. [125I]CsA added to a hemolysate eluted much later in the same position as [3H]CsA mixed with albumin and myoglobin (presumably as free unbound drug). These findings indicate that CsA normally binds to an intraerythrocytic protein similar in molecular size to calf thymus cyclophilin (Mr 15,000). By equilibrium dialysis, the purified erythrocyte proteins calmodulin (Mr 16,700) and cytochrome b5 (Mr 15,000) failed to bind CsA. By equilibrium dialysis, [3H] CsA did bind to column fractions containing the CsA-binding protein, but [125I]CsA did not, suggesting that attachment to CsA occurs at or near a carbon-carbon double bond in an unusual nine-carbon amino acid of CsA. These results have important implications for CsA therapy with regard to distribution space, pharmacokinetics, and a possible protein-receptor mechanism of action.

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.

Serum albumin and the metabolism of disulfiram.

The effectiveness of tetraethylthiuram disulfide (DSF) as a drug used in the treatment of alcohol abuse has been limited by the fact that it is degraded rapidly in the tissues and in the serum. Hence, a useful dose-response curve for this drug cannot be determined easily. The degradation in the tissues has been well characterized; however, its fate in the serum is less well understood. Here we kinetically describe the first steps in the degradation of DSF in the serum which results from a covalent interaction of this drug with the free sulfhydryl of serum albumin. DSF and its cleavage product diethyldithiocarbamate (DDC) both absorb significantly in the ultraviolet region. The reduction of DSF with mercaptoethanol to two molecules of DDC resulted in a large change in absorption in this region. The reaction of serum albumin with DSF produced a similar but much slower change in the ultraviolet absorption. As a result of the existence of this slow spectral change, we have been able to directly and continuously monitor the interaction of serum albumin and DSF and have determined that it is an overall first-order process. A model is proposed wherein DSF and serum albumin rapidly form a noncovalent adduct and, subsequently, in a slow unimolecular process, DSF is reduced to one mole of free DDC and one mole of the serum albumin-DDC mixed disulfide. At pH 9 the half-time for this process was 30 to 40 sec, and at pH 7.4 the half-time for this process was 1 to 1.5 min. These results suggest that degradation of DSF by serum albumin is physiologically and clinically important since the drug is maximally active only many hours after administration.

Thromboembolism in Hageman trait.

The case of a patient with severe deficiency of Hageman factor (factor XII) in whom a thrombus embolized to arteries in a lower extremity is described. In addition to its action in intiating blood clotting through the intrinsic pathway, Hageman factor can influence kinin generation, fibrinolysis, and activation of complement. While individuals with Hageman factor deficiency have no hemorrhagic disorder, this case emphasizes the role factor XII may normally assume in clot dissolution.

Pathogenesis of Pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study.

Pichinde virus has been adapted to produce lethal infection of Strain 13 guinea pigs. Viral replication and presence of viral antigen in frozen tissues stained by immunofluorescence has been previously described. Further investigation into the pathogenesis of this disease has been hampered by the lack of a light microscopic method for correlating histologic lesions and the presence of Pichinde viral antigens. For this purpose, we developed a sensitive immunocytochemical technique for staining Pichinde viral antigens in formalin-fixed, paraffin-embedded tissue. Enhancement of the immunocytochemical staining with nickel chloride markedly improved detection of viral antigens. We examined frozen and formalin-fixed tissues from Strain 13 guinea pigs for viral antigens by light microscopy and immunocytochemistry at various intervals after infection with Pichinde virus. Progressive involvement of different tissues correlated with organ injury measured by serum biochemical abnormalities. Pichinde viral antigen was first detected in splenic macrophages five days after infection and their subsequent destruction facilitated persistent viremia. The inability to clear virus led to multiple organ infection and vascular involvement. Ensuing infections involved particularly the liver, spleen, adrenal glands, lungs, and intestines. Gastroenteritis developed, with extensive involvement of the muscularis mucosa throughout the gastrointestinal tract. Water and food intake decreased rapidly after day 8, leading to marked weight loss. Fatty changes of the liver suggested metabolic derangement that was further exacerbated terminally by adrenal infection and pulmonary impairment.

Daily variability in human serum prostate-specific antigen and prostatic acid phosphatase: a comparative evaluation.

The daily variation of serum levels of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) was investigated simultaneously in 10 patients with osseous metastatic prostatic cancer, 10 patients with benign prostatic hyperplasia, and 10 volunteers without prostatic disease. Duplicate serum samples were obtained from all patients on the same day at 8 AM, 12 PM, 4 PM, and 8 PM. Statistical analysis (two-factor analysis of variance comparing time period to disease group) of the mean PSA and PAP levels at the four sampling times on all patient groups demonstrated no evidence of circadian rhythmic variation or any other distinct pattern for the observed sample times. Overall, the variability in PSA levels was significantly less than that observed for PAP. There was no significant difference in mean percent variation between patient groups (cancer, benign, and normal prostate glands) for both the PSA and PAP assays. Our data reveal that serum PSA measurements fluctuate unpredictably over the course of a day in patients with and without prostatic disease, but to a lesser extent than that seen for serum PAP values. These findings illustrate the potential inaccuracy of single determinations of serum PAP or PSA levels for monitoring disease recurrence and treatment response in patients with prostate cancer.

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.

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.

Serologic evaluation of renal status.

Mechanisms of immunologic injury causing glomerulonephritis are based on immune complex or autoantibody deposition with activation of complement. Circulating immune complex disease can be diagnosed and monitored with improved assays for immune complexes, DNA antibodies, complement levels, and complement activation products. Etiologic auto-antibodies for the Goodpasture antigen, neutrophil enzymes, and ones directly promoting complement activation are now quantitated for more accurate serologic diagnosis. The spectrum of postinfectious glomerulonephritis also presents several new measurements for assessing clinical status. Diagnosis and monitoring of glomerulonephritis now entails panel testing for these humoral abnormalities that are useful to establish the etiology and treatment regimens.

Update of autoantibodies in neurologic disease.

Many neurologic disorders recently have been discovered to have an autoimmune basis with autoantibodies that are responsible for neurologic symptoms and progression of the disease. Diagnosis and treatment of these disorders are facilitated by detection and monitoring of several autoantibodies. Some tumors express protein antigens shared by neurons, and an antitumor immune response leads to autoimmune antineuronal reactions exemplified by "paraneoplastic cerebellar degeneration." Other antibodies react with gangliosides present in many locations of the nervous system, leading to motor neuron damage and polyneuropathy. Phospholipid antibodies indirectly cause neurologic syndrome largely through thrombosis of cerebral vessels; other autoantibodies also have associations with specific neurologic symptoms.

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.

Genetic mutations in ten unrelated American patients with symptomatic type 1 protein C deficiency.

Symptomatic patients with Type 1 protein C deficiency and venous thrombosis were analysed for defects in this gene using polymerase chain reaction amplification and direct sequencing of all nine exons. Ten different heterozygous point mutations were detected in 19 patients from eleven American families. Seven represent novel mutations. Two of these were found in the TATA box or near the transcription initiation site and presumably lead to loss of transcription, and seven missense mutations were found including G103R, P168L, R169W, I201T, P279L, T298M, and C384Y. These may lead to abnormal folding or thermodynamic instability of the protein C molecule, potentially causing abnormal secretion or rapid clearance from the circulation. Two other protein C mutations, a nonsense mutation at codon Trp-145 and a deletion inducing a frameshift at codon 364 resulting in premature termination at codon 378, likely lead to unstable products. The previously published R169W mutation resulted in a Type 1 deficiency. The data show that diverse molecular defects result in similar phenotypes and emphasize that a wide variety of mutations are responsible for Type 1 protein C deficiency in the American setting of a diverse population.

Theoretical analysis of ligand-binder complex formation: partial equilibrium and incubation time.

This report presents a theoretical model of the rate at which complex formation approaches equilibrium in competitive binding systems while ligand concentration varies. Three commonly encountered systems are examined in which binder concentrations (q) are fixed at 0.5/K, 3/K and 50/K, respectively. Our analysis of the incubation time for any particular fraction of reaction completeness shows that longest incubation times occur with the lowest ligand concentrations when q = 0.5/K. However, in systems where q = 3/K and q = 50/K, the longest incubation times correspond not to lowest ligand concentrations but to those approximating the concentration of binder. It is concluded that empirical determination of minimal incubation times for these latter two assay systems should be performed with ligand and binder in approximately equal concentrations.