技术资料

OBJECTIVE: The aim of this study was the preclinical evaluation of imatinib mesylate (Gleevec,formerly STI571) in conjunction with arsenic trioxide (As2O3,Trisenox) for the treatment of chronic myelogenous leukemia (CML). MATERIALS AND METHODS: Tetrazolium-based cell line proliferation assays (MTT assays) were performed to determine the cytotoxicity of As2O3 alone and in combination with imatinib. Cell lines tested in this study were Bcr-Abl-expressing cells (K562,MO7p210,32Dp210) and parental cells (MO7e,32D). Isobologram analysis was performed manually and using the median effect method. In vitro cytotoxicity also was determined in colony-forming assays using CML patient cells. Western blot analysis was performed to detect Bcr-Abl protein levels in K562 cells exposed to As2O3 at graded concentrations. Bcr-Abl protein level kinetics were correlated with cell viability (trypan blue count) and activated caspase-3 detected by flow cytometry. RESULTS: We show additive to synergistic cytotoxicity in Bcr-Abl+ cell lines depending on inhibitory concentrations and cell type. Results obtained by colony-forming assays confirmed the findings in cell line proliferation assays. Flow cytometric detection of activated caspase-3 revealed synergistic activity in K562 cells. Treatment of K562 cells with As2O3 alone led to down-regulation of Bcr-Abl protein within 24 hours,even at low doses. The decline of Bcr-Abl preceded activation of caspase-3 and the loss of viable cells. CONCLUSIONS: Favorable cytotoxicity and proapoptotic activity of imatinib in conjunction with As2O3 and specific down-regulation of Bcr-Abl protein levels by As2O3 in K562 cells indicate that As2O3 in combination with imatinib might be useful for circumventing resistance to imatinib monotherapy. View Publication

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here,we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10,25,or 50 mWcm(-2) and,(1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells,the photobleaching,and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However,in all of the cases,cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT,as well as confirming the likely role of (1)O(2) in porphyrin-based PDT. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Constant deregulation of Id1 and Id3 has been implicated in a wide range of carcinomas. However,underlying molecular evidence for the joint role of Id1 and Id3 in the tumorigenicity of small cell lung cancer (SCLC) is sparse. Investigating the biological significance of elevated expression in SCLC cells,we found that Id1 and Id3 co-suppression resulted in significant reduction of proliferation rate,invasiveness and anchorage-independent growth. Suppressing both Id1 and Id3 expression also greatly reduced the average size of tumors produced by transfectant cells when inoculated subcutaneously into nude mice. Further investigation revealed that suppressed expression of Id1 and Id3 was accompanied by decreased angiogenesis and increased apoptosis. Therefore,the SCLC tumorigenicity suppression effect of double knockdown of Id1 and Id3 may be regulated through pathways of apoptosis and angiogenesis. View Publication

Human B cells can be cultured ex vivo for a few weeks,following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However,attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells,recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4,the main LPS receptor,prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells,accompanied by an increase in antigen-specific antibody secretion. This result suggested that some,but not all,B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6,a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo,the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria,until all pathogens have been cleared from the organism. View Publication

HOX genes,notably members of the HOXA cluster,and HOX cofactors have increasingly been linked to human leukemia. Intriguingly,HOXD13,a member of the HOXD cluster not normally expressed in hematopoietic cells,was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months),when some mice developed a myeloproliferative-like disease. In contrast,mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML),with a median disease onset of 75 days. In summary,this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation,and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication

PURPOSE: The propensity of tumor cells to escape immune elimination could limit,if not defeat,the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is,however,not known whether T cells corresponding to stromal products are present in humans. In this study,we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens,we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products,which are overexpressed in the tumor stroma,are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product,a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP,a protease preferentially expressed in tumor-associated fibroblasts,as a candidate human stromal antigen to target in the setting of cancer immunotherapy,and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting. View Publication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia,an aggressive CD4(+) malignancy. Although HTLV-2 is highly homologous to HTLV-1,infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34(+) cells with HTLV-1 Tax (Tax1) induced G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (p27),and marked suppression of hematopoiesis in immature (CD34(+)/CD38(-)) hematopoietic progenitor cells in comparison to CD34(+)/CD38(+) cells. HTLV-1 infection of CD34(+) cells also induced p21 and p27 expression. Tax1 also protected CD34(+) cells from serum withdrawal-mediated apoptosis. In contrast,HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression,failed to induce cell cycle arrest,failed to suppress hematopoiesis in CD34(+) cells,and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax(221)) displayed a phenotype in CD34(+) cells similar to that of Tax1,suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34(+) hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication

A commonly activated signaling cascade in many human malignancies,including glioblastoma multiforme,is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor,PTEN deletion,or PIK3CA mutations. In this study,we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule,A-443654,showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings,this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654,methylated at a region that blocks Akt binding,was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion,respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma,with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions. View Publication

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions,including cell cycle progression; cell migration,invasion,and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene,which is located downstream of PI3K,have been shown in a variety of cancers,including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways,including inhibitors of the PI3K pathway,have shown antitumor activity,but inhibitors of Akt have not been examined. In this study,we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors,KP-372-1 and KP-372-2 (herein called KP-1 and KP-2),effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L,which,in turn,reduced the activation of intracellular downstream targets of Akt,including GSK-3beta and p70s6k. Furthermore,the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively,these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas. View Publication