Scientific Resources

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses,different therapeutic approaches have been investigated. Recently,the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful,it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents,comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism,since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro,and as many activities may be present in solid tumours,including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially,DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity,this does not hold consistently in vivo for any single bioreductive enzyme,suggesting revision of the enzyme-directed hypothesis as originally formulated. View Publication

Phosphatidylinositol,a component of eukaryotic cell membranes,is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol,collectively termed phosphoinositides,have been identified in eukaryotic cells from yeast to mammals. Phosphoinositides are involved in the regulation of diverse cellular processes,including proliferation,survival,cytoskeletal organization,vesicle trafficking,glucose transport,and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphoinositide kinases. Recent advances have challenged previous hypotheses about the substrate selectivity of different phosphoinositide kinase families. Here we re-examine the pathways of phosphoinositide synthesis and the enzymes involved. View Publication

We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase,and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus,r = 0.972 for E. coli,and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 microg/ml for S. aureus topoisomerase IV; IC50 = 0.109 microg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 microg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However,the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones,indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases. View Publication

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here,we identify a newly discovered human repopulating cell,distinct from previously identified repopulating cells,that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells,or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR,Thy-1 and CD34). In contrast to CD34+ subfractions,Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations,providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation. View Publication

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437),originally identified as a retinoic acid receptor gamma-selective retinoid,was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study,we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis,a process that is accompanied by rapid induction of c-Jun,nur77,and p21(WAF1/CIP1). In addition,we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein,a dominant-negative inhibitor of c-Jun,in A549 cells,nur77 expression and apoptosis induction by AHPN/CD437 were impaired,whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore,overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus,expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together,our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer. View Publication

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 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

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

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

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3),IL-6,granulocyte colony-stimulating factor,Flt3-ligand,and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells,colony-forming cells (CFC),or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period,and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However,the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant,17% +/- 3% and 17% +/- 8%,G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected,the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM),or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17,respectively),whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a textgreater/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols. View Publication