技术资料

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

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore,one stem cell might easily account for the renewal of mammary epithelium over several transplant generations. View Publication

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations,we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases,including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation,inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound,the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase,we also showed that cells expressing an activated PDGFR tyrosine kinase,TEL-PDGFR,are sensitive to this compound. Thus,this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein. View Publication

The effect of 9-cis-retinoic acid (9-cis-RA) on the growth of eight gastric cancer cell lines was related to their transcription levels of mRNAs for retinoid receptors. Northern blot analysis showed that seven (TMK-1,MKN-1,-28,-45,-74,HSC-39,KATO-III) out of eight gastric cancer cell lines synthesized mRNAs for retinoic acid receptors (RARs) and retinoid X receptor-alpha (RXR-alpha). MKN-7 cells did not transcribe either RARs or RXR-alpha at the mRNA level although they appeared to have no alterations at the gene level. The growth of all of the cell lines except for MKN-7 cells was inhibited by 1 x 10(-6) M 9-cis-RA. Cell cycle distribution analysis revealed that G0-G1 arrest was not induced by exposure to 9-cis-RA in the sensitive TMK-1 and KATO-III cells or the resistant MKN-7 cells. Interestingly,9-cis-RA temporarily increased the amount of the cyclin dependent kinase (cdk) inhibitor,Waf1/Cip1/Sdi1/p21 protein,and also reduced the amount of cdk-7,epidermal growth factor receptor (EGFR) and cyclin D1 proteins,followed by reduction in phosphorylation of the product of the retinoblastoma tumor suppressor gene (Rb) in the sensitive TMK-1 cells,but not in the resistant MKN-7 cells. These results suggest that 9-cis-RA has a cytostatic effect on gastric cancer cells that synthesize the receptor molecules through cell cycle regulatory machinery. View Publication

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study,we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs),since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity,but 9-cis-retinoic acid (RA),13-cis-RA,and all-trans-RA (ATRA) were markedly more efficacious than Ro40-8757,Ro13-6298,and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs,thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1,a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems,the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact,retinoid-induced modifications of cell morphology,phenotype (downregulation of CD19,HLA-DR,and s-Ig,and increased expression of CD38 and c-Ig),and IgM production were late events,highly heterogeneous,and often slightly relevant,being therefore only partially indicative of a drug-related differentiative process. Moreover,EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids,indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

BACKGROUND: At therapeutic concentrations,the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis,similar to the mechanism of cell death induced by other antineoplastic agents. However,taxol has shown efficacy against drug-refractory cancers,raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block,aberrant mitosis,and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or,for those cells progressing through aberrant mitosis,arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast,G1-arrested,multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase,both wt p53-expressing and p53-null MEFs responded similarly to taxol,showing rapid onset of DNA nicking and apoptosis. However,p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive,and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest,which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway,whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent. View Publication

The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM,however,resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,000 nM) had an increase in survival compared with cells treated with lower concentrations of the drug. Prolonging the time of exposure of cells to paclitaxel from 24 to 72 h increased cytotoxicity from 5 to 200 fold in different cell lines. Exponentially growing cells were more sensitive to paclitaxel than were cells in the plateau phase of growth. Cremophor EL,the diluent in which the clinical preparation of paclitaxel is formulated,antagonised paclitaxel at concentrations of 0.135% (v/v). These data suggest that paclitaxel will be most effective clinically when there is prolonged exposure of tumour to the drug. Further,it appears that modest concentrations (i.e.,50 nM) should be as effective as higher concentrations of paclitaxel. Finally,we have noted that Cremophor EL is a biologically active diluent and,at high concentrations (0.135% v/v),can antagonise paclitaxel cytotoxicity. View Publication

The microbial product wortmannin has previously been shown to be a potent inhibitor of phosphatidylinositol-3-kinase. In view of the potential role of this enzyme in transduction of mitogenic signals,we determined the cytotoxic activity of wortmannin against several human tumor cell lines in vitro. The most sensitive lines included GC3 colon carcinoma,IGROV1 ovarian carcinoma,and CCRF-CEM leukemia (IC-50s ranging from 0.7-2.1 microM). The cytotoxicity of wortmannin was decreased approximately 10-fold by serum-free conditions. Wortmannin was generally less active in low passage human breast cancer cell lines that overexpress either epidermal growth factor receptor or Her2/neu. Wortmannin was also tested for in vivo antitumor activity against seven murine tumor and ten human tumor xenograft models. Activity (textgreater 60% inhibition of tumor growth) was observed in only the C3H mammary carcinoma and the human BxPC-3 pancreatic carcinoma xenograft. In vivo antitumor activity did not correlate with in vitro sensitivity to wortmannin cytotoxicity. View Publication

Taxol stabilizes microtubules,prevents tubulin depolymerization,and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized,the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein,we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells,although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells,wild-type p53 protein was also induced after taxol treatment,and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells,p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects,taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol,as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity,but that it is not strictly dependent on wild-type p53. Furthermore,the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression. View Publication

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system,an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points,CTCs were detected in 37{\%},54.9{\%} and 58.7{\%} of patients using CellSearch,CellCollector and EPISPOT,respectively. The cumulative positivity rate of the three CTC assays was 81.3{\%} (87/107) with 21.5{\%} (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66{\%} before RP to 34{\%} after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p {\textless} 0.0001) and clinical tumor stage (p = 0.04),while the other assays showed no significant correlations. In conclusion,CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer. View Publication