Effective mass anisotropy of hot electrons in nonparabolic conduction bands of n-doped InGaAs films using ultrafast terahertz pump-probe techniques.

The anisotropic effective mass of energetic electrons in an isotropic, nonparabolic conduction band is revealed using ultrafast THz-pump-THz-probe techniques in a n-doped InGaAs semiconductor thin film. A microscopic theory is applied to identify the origin of the observed anisotropy and to show that the self-consistent light-matter coupling contributes significantly to the THz response.

Fano signatures in the intersubband terahertz response of optically excited semiconductor quantum wells.

Absorption and transmission spectra of broadband terahertz pulses are measured to probe the intersubband response of an optically excited quantum-well heterostructure. While the terahertz absorption shows the single peak of the resonant intersubband transition, the transmission spectra display strong Fano signatures due to the phase sensitive superposition of ponderomotive and terahertz currents as predicted by our microscopic theory.

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells.

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.

Dehydroascorbic acid, a blood-brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke.

Neuronal injury in ischemic stroke is partly mediated by cytotoxic reactive oxygen species. Although the antioxidant ascorbic acid (AA) or vitamin C does not penetrate the blood-brain barrier (BBB), its oxidized form, dehydroascorbic acid (DHA), enters the brain by means of facilitative transport. We hypothesized that i.v. DHA would improve outcome after stroke because of its ability to cross the BBB and augment brain antioxidant levels. Reversible or permanent focal cerebral ischemia was created by intraluminal middle cerebral artery occlusion in mice treated with vehicle, AA, or DHA (40, 250, or 500 mg/kg), either before or after ischemia. Given before ischemia, DHA caused dose-dependent increases in postreperfusion cerebral blood flow, with reductions in neurological deficit and mortality. In reperfused cerebral ischemia, mean infarct volume was reduced from 53% and 59% in vehicle- and AA-treated animals, respectively, to 15% in 250 mg/kg DHA-treated animals (P < 0.05). Similar significant reductions occurred in nonreperfused cerebral ischemia. Delayed postischemic DHA administration after 15 min or 3 h also mediated improved outcomes. DHA (250 mg/kg or 500 mg/kg) administered at 3 h postischemia reduced infarct volume by 6- to 9-fold, to only 5% with the highest DHA dose (P < 0.05). In contrast, AA had no effect on infarct volumes, mortality, or neurological deficits. No differences in the incidence of intracerebral hemorrhage occurred. Unlike exogenous AA, DHA confers in vivo, dose-dependent neuroprotection in reperfused and nonreperfused cerebral ischemia at clinically relevant times. As a naturally occurring interconvertible form of AA with BBB permeability, DHA represents a promising pharmacological therapy for stroke based on its effects in this model of cerebral ischemia.

The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.

The glucose transporter GLUT1 has three short amino acid sequences (domains I-III) with homology to typical ATP-binding domains. GLUT1 is a facilitative transporter, however, and transports its substrates down a concentration gradient without a specific requirement for energy or hydrolysis of ATP. Therefore, we assessed the functional role of the predicted ATP-binding domains in GLUT1 by site-directed mutagenesis and expression in Xenopus oocytes. For each mutant, we determined the level of protein expression and the kinetics of transport under zero-trans influx, zero-trans efflux, and equilibrium exchange conditions. Although all five mutants were expressed at levels similar to that of the wild-type GLUT1, each single amino acid change in domains I or III profoundly affected GLUT1 function. The mutants Gly116-->Ala in domain I and Gly332-->Ala in domain III exhibited only 10-20% of the transport activity of the wild-type GLUT1. The mutants Gly111-->Ala in domain I and Leu336-->Ala in domain III showed altered kinetic properties; neither the apparent Km nor the Vmax for 3-methylglucose transport were increased under equilibrium exchange conditions, and they did not show the expected level of countertransport acceleration. The mutant Lys117-->Arg in domain I showed a marked increase in the apparent Km for 3-methylglucose transport under zero-trans efflux and equilibrium exchange conditions while maintaining countertransport acceleration. These results indicate that the predicted ATP-binding domains I and III in GLUT1 are important components of the region in GLUT1 involved in transport of the substrate and that their integrity is critical for maintaining the activity and kinetic properties of the transporter.

Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors.

The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.

Critical evaluation of the World Health Organization classification of myelodysplasia and acute myeloid leukemia.

The separation of myelodysplastic syndromes from acute myeloid leukemia has been problematic because, as clinical entities, they represent different points in the spectrum of the same disease process. A new classification by the World Health Organization has incorporated recent prognostic findings in myelodysplastic syndrome and distinct genetic subtypes of acute myelogenous leukemia to provide an improved conceptual framework, if not a simpler nomenclature. Here, we review the new classification system and discuss its impact on diagnosis and treatment.

Consolidation therapy with high-dose cyclophosphamide improves the quality of response in patients with chronic lymphocytic leukemia treated with fludarabine as induction therapy.

Fludarabine is the most active agent in the treatment of chronic lymphocytic leukemia (CLL). Despite this activity only a minority of patients treated with fludarabine achieve a complete response. We evaluated a new treatment program of sequential therapy with fludarabine followed by high-dose cyclophosphamide in previously untreated patients with CLL. This report details the results in 25 patients with previously untreated CLL. Patients received fludarabine (25 mg/m2/day x 5 days every 4 weeks for six cycles) as induction followed by consolidation with high-dose cyclophosphamide at one of three dose levels 1.5 g/m2, 2.25 g/m2, or 3 g/m2 administered every 2 weeks for three doses. High-dose cyclophosphamide was given with G-CSF support (5 microg/kg/day days 3-12). Complete response (CR) required a normal physical examination, normal CBC, a normal bone marrow evaluation including no residual lymphoid nodules on biopsy. A nodular response was defined as a complete response with the exception of an occasional residual nodule seen on bone marrow biopsy. Flow cytometric analysis for CD5:CD19 dual staining and kappa/lambda clonal excess was performed in all patients as a sensitive measure of minimal residual disease (MRD). Selected patients had patient/tumor-specific oligonucleotides generated that were subsequently used in a polymerase chain reaction as an extremely sensitive measure of MRD. There were no treatment-related deaths and no patient encountered unacceptable toxicity. After completion of this sequential regimen 76% (95% confidence interval: 59-93%) of patients had a major response: eight (32%) achieved a CR, four (16%) a nodular response, seven (28%) a PR, and six patients (24%) failed. Four patients withdrew from study during induction with fludarabine and did not receive at least one cycle of cyclophosphamide. Of the 21 patients who received consolidation with cyclophosphamide 10 (48%) had an improved quality of response when compared to that achieved with fludarabine. Two patients (8%) had no disease detectable by flow cytometry ('flow cytometric' CR) after six cycles of fludarabine. This improved to nine patients (36%) after high-dose cyclophosphamide. Following consolidation with high-dose cyclophosphamide three patients (12%) tested negative by PCR. All of these patients had morphologic evidence of residual disease after six cycles of fludarabine. Consolidation with high-dose cyclophosphamide increased the fraction of patients achieving a nodular response or CR three-fold (16% to 48%). This appears to be clinically relevant because with a median follow-up of 52 (range 34-78) months the projected 6-year survival for patients achieving a CR or NR is 91% compared to 41% for all others (P = 0.012). We conclude that sequential therapy with fludarabine followed by high-dose cyclophosphamide in previously untreated patients with CLL is safe and can improve the quality of response in a large proportion of patients compared to therapy with fludarabine alone.

Molecular characterization of a granulocyte macrophage-colony-stimulating factor receptor alpha subunit-associated protein, GRAP.

The granulocyte macrophage-colony-stimulating factor receptor (GM-CSF-R) is a heterodimer composed of 2 subunits, alpha and beta, and ligand binding to the high-affinity receptor leads to signalling for the multiple actions of GM-CSF on target cells. In order to explore the role of the alpha subunit in signalling, we used a yeast-2-hybrid system to identify proteins interacting with the intracellular domain of the GMR-alpha. A cDNA encoding a predicted protein of 198 amino acids, designated GRAP (GM-CSF receptor alpha subunit-associated protein), was isolated in experiments using the intracellular portion of GMR-alpha as bait. The interaction between GRAP and GMR-alpha was confirmed by coimmunoprecipitation in mammalian cells. GRAP mRNA is widely expressed in normal human and mouse tissues and in neoplastic human cell lines, but it is not restricted to cells or tissues that express GM-CSF receptors. Three discrete GRAP mRNA species were detected in human tissues and cells, with estimated sizes of 3.3, 3.1, and 1.3 kb. GRAP is highly conserved throughout evolution, and homologues are found in yeast. The GRAP locus in Saccharomyces cerevisiae was disrupted, and mutant yeast cells showed an inappropriate stress response under normal culture conditions, manifested by early accumulation of glycogen during the logarithmic growth phase. GRAP is, therefore, a highly conserved and widely expressed protein that binds to the intracellular domain of GMR-alpha, and it appears to play an important role in cellular metabolism.

High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the beta subunit.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMRalpha and GMRbeta. GMRalpha in isolation binds to GM-CSF with low affinity. GMRbeta does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMRalpha. Previously, it was found that N-glycosylation of GMRalpha is essential for ligand binding. The present study investigated the role of N-glycosylation of the beta subunit on GM-CSF receptor function. GMRbeta has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRbeta mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRbeta in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.

Prostate cancer cell cycle regulators: response to androgen withdrawal and development of androgen independence.

BACKGROUND: Androgen withdrawal is a standard therapy for prostate cancer that results in a decrease in tumor volume and a decline in serum prostate-specific antigen in the majority of patients. To understand the factors associated with regression of prostate cancers after androgen withdrawal, we studied cell cycle regulator changes in the CWR22 human prostate cancer xenograft model. METHODS: Established tumors in nude athymic BALB/c mice were sampled at various times after androgen withdrawal and after the development of androgen independence. Changes in the expression of cell cycle regulators were categorized into early and mid-to-late events. RESULTS AND CONCLUSIONS: Early events included a decrease in androgen receptor expression, followed by a short-term increase in expression of the p53 and p21/WAF1 proteins and a marked decrease in the Ki67 proliferative index. Mid-to-late events included progressive and sustained increases in p27 and p16 protein expression, a decrease in retinoblastoma protein expression, and an increase in the transcription factor E2F1. Changes in apoptosis (programmed cell death) were not observed at any time after androgen withdrawal. These data suggest that androgen withdrawal results in a cell stress response, in which increased p53 protein produces a cell cycle arrest, without activation of p53-mediated apoptosis. The proliferative index is further decreased through the action of the cyclin-dependent kinase inhibitors p27 and p16. Androgen-independent sublines emerged 80-400 days after androgen withdrawal, and these sublines had variable growth phenotypes but were associated with mdm2 protein overexpression and increased expression of cyclin D1. These results indicate that tumor regression in this human prostate cancer model is due to cell cycle arrest rather than to apoptosis and that the emergence of androgen independence is associated with a release from cell cycle arrest.

Kinetic resolution of two mechanisms for high-affinity granulocyte-macrophage colony-stimulating factor binding to its receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMRalpha, which binds GM-CSF with low affinity, and GMRbeta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (GMRalpha/beta). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that GMRalpha/beta exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMRalpha/beta points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.

Response of prostate cancer to anti-Her-2/neu antibody in androgen-dependent and -independent human xenograft models.

Antibody to the Her-2/neu gene product has been shown to inhibit the growth of breast cancer cells overexpressing Her-2/neu and to have clinical utility in treating breast cancer. We studied a recombinant, humanized anti-Her-2/neu antibody (Herceptin) in preclinical models of human prostate cancer. The androgen-dependent CWR22 and LNCaP human prostate cancer xenograft models and androgen-independent sublines of CWR22 were used. Her-2/neu staining of the parental, androgen-dependent, and androgen-independent CWR22 tumors and LNCaP tumors demonstrated variable Her-2/neu expression. Herceptin was administered i.p. at a dose of 20 mg/kg twice weekly after the xenograft had been established. No effect of Herceptin on tumor growth was observed in any of the androgen-independent tumors; however, significant growth inhibition was observed in both of the androgen-dependent xenograft models, CWR22 (68% growth inhibition at the completion of the experiment; P = 0.03 for trajectories of the average tumor volume of the groups) and LNCaP (89% growth inhibition; P = 0.002). There was a significant increase in prostate-specific antigen (PSA) index (ng PSA/ml serum/mm3 tumor) in Herceptin-treated androgen-dependent groups compared with control (CWR22, 18-fold relative to pretreatment value versus 1.0-fold, P = 0.0001; LNCaP, 2.35-fold relative to pretreatment value versus 0.6-fold, P = 0.001). When paclitaxel (6.25 mg/kg s.c., five times/week) was given to animals with androgen-dependent and -independent tumors, there was growth inhibition in each group. Paclitaxel and Herceptin cotreatment led to greater growth inhibition than was seen for the agents individually. Thus, in these prostate cancer model systems, Herceptin alone has clinical activity only in the androgen-dependent tumor and has at least an additive effect on growth, in combination with paclitaxel, in both androgen-dependent and androgen-independent tumors. Response to Herceptin did not correlate with the PSA levels, because the PSA index markedly increased in the Herceptin-treated group, whereas it remained constant in the control group. These results suggest the utility of Herceptin in the treatment of human prostate cancer.

Stromal cell oxidation: a mechanism by which tumors obtain vitamin C.

Human tumors may contain high concentrations of ascorbic acid, but little is known about how they acquire the vitamin. Certain specialized cells can transport ascorbic acid directly through a sodium ascorbate cotransporter, but in most cells, vitamin C enters through the facilitative glucose transporters (GLUTs) in the form of dehydroascorbic acid, which is then reduced intracellularly and retained as ascorbic acid. Mice with established hematopoietic and epithelial cell xenografts were studied for the accumulation of injected ascorbic acid and dehydroascorbic acid. Most hematopoietic and epithelial tumor cell lines can only transport vitamin C in the oxidized form (dehydroascorbic acid) in vitro; however, when grown as xenografts in mice, they rapidly accumulated vitamin C after administration of radiolabeled ascorbic acid. The involvement of the GLUTs in vitamin C uptake by the xenografted tumors was demonstrated by competitive inhibition with D-glucose but not L-glucose. Because the malignant cells were not capable of directly transporting ascorbic acid, we reasoned that the ascorbic acid was oxidized to dehydroascorbic acid in the tumor microenvironment. Tumor accumulation of vitamin C in animals injected with ascorbic acid was inhibited by coadministration of superoxide dismutase, implying a role for superoxide anion in the oxidation of ascorbic acid. Whereas the epithelial cancer cell lines could not generate superoxide anion in culture, the minced xenograft tumors did. Our studies show the transport of dehydroascorbic acid by GLUTs is a means by which tumors acquire vitamin C and indicate the oxidation of ascorbic acid by superoxide anion produced by cells in the tumor stroma as a mechanism for generating the transportable form of the vitamin.

Expression of the human GM-CSF receptor alpha subunit in Saccharomyces cerevisiae.

The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation. Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction. To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S. cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells. Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane. Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF. Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.

Human monocarboxylate transporter 2 (MCT2) is a high affinity pyruvate transporter.

The transport of pyruvate and lactate across cellular membranes is an essential process in mammalian cells and is mediated by the H+/monocarboxylate transporters (MCTs). We have molecularly cloned and characterized a novel human monocarboxylate transporter, MCT2. The cDNA is 1,907 base pairs long and encodes a polypeptide of 478 amino acids with 12 predicted transmembrane domains. Human MCT2 is the product of a single gene that mapped to chromosome 12q13 by fluorescence in situ hybridization. The kinetic properties of human MCT2 fulfill the criteria to establish it as a H+/monocarboxylate transporter; however, the unique biochemical feature of human MCT2 is its high affinity for the transport of pyruvate (apparent Km of 25 microM), implying that it is a primary pyruvate transporter in man. Comparison of human MCT1 and MCT2 with regard to tissue distribution and RNA transcript variants disclosed substantial differences. Human MCT2 mRNA expression was restricted in normal human tissues but widely expressed in cancer cell lines, suggesting that MCT2 may be pre-translationally regulated in neoplasia. We found co-expression of human MCT1 and MCT2 at the mRNA level in human cancer cell lines, including the hematopoietic lineages HL60, K562, MOLT-4, and Burkitt's lymphoma Raji, and solid tumor cells such as SW480, A549, and G361. These findings suggest that the two monocarboxylate transporters, MCT1 and MCT2, have distinct biological roles.

Soluble receptors in human disease.

Soluble cytokine receptors naturally arise from genes encoding membrane-bound receptors or are direct derivatives of the receptors themselves. There is mounting evidence that soluble receptors play important roles in human disease states. In many cases, soluble receptors appear to play an integral part in the dynamic interaction between ligands and their membrane-bound receptors in maintaining and restoring health after a pathological insult but, in some instances, dysregulated expression of soluble receptors can contribute to disease pathology. Nonetheless, an appreciation of the biological actions of soluble receptors, particularly as cytokine inhibitors, has led to their therapeutic use in human diseases. Although early clinical trails of soluble receptors have had unexpected toxicities, their application in medicine continues to advance and it is likely that soluble receptors will join hormones, cytokines, and growth factors as established biological therapies.

Positron emission tomography of a human prostate cancer xenograft: association of changes in deoxyglucose accumulation with other measures of outcome following androgen withdrawal.

The current means of assessing response in prostate cancer are imprecise because changes in tumor size are often slow and difficult to document, and alterations in serum prostate-specific antigen (PSA) levels do not always correlate with clinical outcomes. Using the CWR22 human prostate cancer xenograft model, serial changes in prostate tumor metabolism were assessed after androgen withdrawal by [3H]deoxyglucose (DOG) accumulation in the tumors and noninvasively using positron emission tomography (PET) with 2-[18F]-fluoro-2-deoxy-D-glucose as a radiotracer. A significant decrease in [3H]DOG accumulation was observed at 48 h after androgen withdrawal [62% of preandrogen withdrawal value (95% confidence interval: 0.59, 0.65)], reaching a maximum decline at day 10 [38% of preandrogen withdrawal value (95% confidence interval: 0.37, 0.40)]. Using PET, parallel changes in tumor metabolism were demonstrated and preceded changes in tumor volume and marked declines in serum PSA. DOG accumulation returned to near baseline upon reintroduction of androgen. The decrease in DOG accumulation was associated with a decline in the proportion of tumor cells in active cell cycle from >60% to <5% at 7-10 days after androgen withdrawal. No increase in the proportion of tumor cells undergoing apoptosis was observed during this time period, implying an arrest in a G0/early G1 state. DOG accumulation in tumor cells, measured directly and by PET, correlated with androgen changes in the host. The results suggest that serial monitoring of DOG accumulation using PET scanning may be useful as an early indicator of treatment efficacy and other outcome measures in prostate cancer.