Scientific Resources

The CD34 molecule belongs to the mucin membrane molecule family and is expressed on virtually all normal haematopoietic progenitor cells (HPC). Due to its heavy glycosylation,several different epitopes exist on the molecule. Based on the sensitivity of the glycosylated molecule to degradation with a glycoprotease from Pasteurella haemolytica and neuraminidase,three classes of epitopes have been identified. The class I and II epitopes are probably related to the glycosylated part of the molecule while class III epitopes are core protein related. It has been known for some time that CD34 class I epitopes are absent on CD34 molecules expressed on high endothelial venules. Here we review recent observations that expression of both class I and II epitopes,but not class III epitopes,is impaired on mature myeloid CD34-pos. HPC while no diverse class epitope expression was observed on immature HPC. In addition,cells from patients with CD34-pos. acute myeloid leukaemia of FAB classification M4-M5,i.e.,leukaemic blast cells of relatively mature morphologic phenotype,also express less class I and II epitopes than class III epitopes. It therefore seems that HPC maturation and class I and II epitope deprivation are concomitant events and that CD34 class I and II epitopes are lost prior to downregulation of the CD34 molecule per se. The biological significance of this observation is discussed as well as the need to carefully select CD34-specific monoclonal antibodies for research and clinical purposes. View Publication

This study sought to examine the relationship between MUC1 expression at the deepest invasive portion,invasive/metastatic potential,and prognosis of colorectal cancer in relation to cellular proliferation. MUC1 expression was detected immunohistochemically using KL-6 antibody (anti-MUC1 monoclonal antibody) in 100 surgically resected specimens of advanced colorectal cancer. Distinct staining of the luminal surfaces,defined as positive immunoreactive (IR)-MUC1 expression,was seen in more than 30% of the tumor cells at the deepest invasive portion. The proliferating cell nuclear antigen labeling index (PCNA-LI) was also examined in the same areas. IR-MUC1 expression was detected in 71 (71%) of 100 lesions. Lesions with lymphatic or venous invasion showed a significantly higher incidence of IR-MUC1 expression than those without lymphatic or venous invasion (80 vs. 42% and 82 vs. 61%,respectively). Lesions with lymph node metastasis showed a significantly higher incidence of IR-MUC1 expression than those without lymph node metastasis (88 vs. 53%). Lesions with liver metastasis showed a significantly higher incidence of IR-MUC1 expression than those without liver metastasis (92 vs. 59%). Dukes' stage was also significantly correlated with IR-MUC1 expression. The incidence of IR-MUC1 expression did not significantly differ with regard to histologic subclassification and depth of invasion. There was no significant correlation between IR-MUC1 expression and the PCNA-LI. IR-MUC1 expression at the deepest invasive portion revealed a significant correlation with prognosis; furthermore,in patients with better differentiated lesions,in those with lesions confined to muscularis propria or subserosa (subadventitial) invasion,in those with Dukes' B and C,or in those undergoing curative resection,IR-MUC1 expression significantly correlated with prognosis. Patients with high PCNA-LI lesions showed a significantly poorer prognosis than those with low PCNA-LI lesions. Only in patients undergoing curative resection,patients with IR-MUC1-positive and high PCNA-LI lesions showed a significantly poorer prognosis than those with IR-MUC1-negative and low PCNA-LI lesions. The significant risk factors in the order of poorer prognosis in patients undergoing curative resection by the multivariate analysis were the histologic grade (moderately-poorly,poorly or mucinous adenocarcinomas),IR-MUC1 expression,and lymph node metastasis. These results indicate that IR-MUC1 expression is an important predictor of the metastatic potential and the prognosis of colorectal cancer,independent of histologic grade,depth of invasion or cellular proliferative activity. Combined analysis of IR-MUC1 and histologic grade,and combined expression of IR-MUC1 and PCNA at the deepest invasive portion are especially useful in predicting colorectal cancer prognosis. View Publication

The compound U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members,MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2,as U0126 shows little,if any,effect on the kinase activities of protein kinase C,Abl,Raf,MEKK,ERK,JNK,MKK-3,MKK-4/SEK,MKK-6,Cdk2,or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley,D. T.,Pang,L.,Decker,S. J.,Bridges,A. J.,and Saltiel,A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92,7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates,ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion,suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly,we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126,its selectivity for MEK over other kinases,and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction. View Publication

The antioxidant properties of butein,isolated from Dalbergia odorifera T. Chen,were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50,3.3+/-0.4 microM. It was as potent as alpha-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0.200,9.2+/-1.8 microM. It also inhibited the activity of xanthine oxidase with an IC50,5.9+/-0.3 microM. Besides,butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase,but not that from 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore,butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL),as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations,and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation. View Publication

A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates,including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats,the two highly related proteins,Stat5a and Stat5b,are activated by a variety of cytokines. To assess the role of the Stat5 proteins,mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential,and often redundant,role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely,the responses to a variety of cytokines that activate the Stat5 proteins,including erythropoietin,are largely unaffected. View Publication

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines,causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified textgreater 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM,whereas no cytotoxicity against EGF-R-negative leukemia cells was observed,even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival,19 days) and all control mice had tumors that rapidly progressed to reach an average size of textgreater 500 mm3 by 58 days,40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size textgreater 50 mm3. Thus,targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. View Publication

Ubiquinone and alpha-lipoic acid are natural constituents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of alpha-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme complexes involved in the citric acid cycle while UQ recycles through semi- and divalently reduced ubiquinones in the respiratory chain. We have proved the validity of the concept about the antioxidant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation,the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product alpha-lipoic acid. Our experiments demonstrate the existence of an dihydrolipoic acid driven recycling of UQ to the antioxidative-active UQH2. The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites. View Publication

BACKGROUND There have been many randomised trials of adjuvant tamoxifen among women with early breast cancer,and an updated overview of their results is presented. METHODS In 1995,information was sought on each woman in any randomised trial that began before 1990 of adjuvant tamoxifen versus no tamoxifen before recurrence. Information was obtained and analysed centrally on each of 37000 women in 55 such trials,comprising about 87% of the worldwide evidence. Compared with the previous such overview,this approximately doubles the amount of evidence from trials of about 5 years of tamoxifen and,taking all trials together,on events occurring more than 5 years after randomisation. FINDINGS Nearly 8000 of the women had a low,or zero,level of the oestrogen-receptor protein (ER) measured in their primary tumour. Among them,the overall effects of tamoxifen appeared to be small,and subsequent analyses of recurrence and total mortality are restricted to the remaining women (18000 with ER-positive tumours,plus nearly 12000 more with untested tumours,of which an estimated 8000 would have been ER-positive). For trials of 1 year,2 years,and about 5 years of adjuvant tamoxifen,the proportional recurrence reductions produced among these 30000 women during about 10 years of follow-up were 21% (SD 3),29% (SD 2),and 47% (SD 3),respectively,with a highly significant trend towards greater effect with longer treatment (chi2(1)=52.0,2ptextless0.00001). The corresponding proportional mortality reductions were 12% (SD 3),17% (SD 3),and 26% (SD 4),respectively,and again the test for trend was significant (chi2(1) = 8.8,2p=0.003). The absolute improvement in recurrence was greater during the first 5 years,whereas the improvement in survival grew steadily larger throughout the first 10 years. The proportional mortality reductions were similar for women with node-positive and node-negative disease,but the absolute mortality reductions were greater in node-positive women. In the trials of about 5 years of adjuvant tamoxifen the absolute improvements in 10-year survival were 10.9% (SD 2.5) for node-positive (61.4% vs 50.5% survival,2ptextless0.00001) and 5.6% (SD 1.3) for node-negative (78.9% vs 73.3% survival,2ptextless0.00001). These benefits appeared to be largely irrespective of age,menopausal status,daily tamoxifen dose (which was generally 20 mg),and of whether chemotherapy had been given to both groups. In terms of other outcomes among all women studied (ie,including those with ER-poor" tumours)� View Publication

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine,by virtue of its polyphenolic constituents,may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene),a polyphenol present in most red wines,on functional and biochemical responses of PMN,upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM,mean+/-s.e.mean),as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM,IC50 18.4+/-1.8 and 31+/-1.8 microM),and C5a (0.1 microM,IC50 41.6+/-3.5 and 42+/-8.3 microM),and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4),6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation,as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb,recognizing an activation-dependent epitope on MAC-1. Consistently,PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results,indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions,may provide some biological plausibility to the protective effect of red wine consumption against CHD. View Publication

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes,the extracellular signal-regulated kinase (ERK),Jun NH2-terminal kinase,and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation,we used the inhibitor U0126,which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK),the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs,but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy,unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly,induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK,unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term,but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants. View Publication

In the present study,we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986),and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms,we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity,lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice,the only effect observed was a cellular infiltration at the site of injection,confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein. View Publication

Butein,a plant polyphenol,was shown to be a specific protein tyrosine kinase inhibitor. This compound inhibited not only the epidermal growth factor (EGF)-stimulated auto-phosphotyrosine level of EGF receptor in HepG2 cells but also tyrosine-specific protein kinase activities of EGF receptor (IC50 = 65 microM) and p60c-src (IC50 = 65 microM) in vitro. The inhibition was competitive to ATP and non-competitive to the phosphate acceptor,poly (Glu,Ala,Tyr) 6:3:1 for EGF receptor tyrosine kinase. In contrast,butein non-significantly inhibited the activities of serine- and threonine-specific protein kinases,such as protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). View Publication