技术资料

Therapeutic mAbs that target tumor-associated Ags on the surface of malignant cells have proven to be an effective and specific option for the treatment of certain cancers. However,many of these protein markers of carcinogenesis are not expressed on the cells' surface. Instead these tumor-associated Ags are processed into peptides that are presented at the cell surface,in the context of MHC class I molecules,where they become targets for T cells. To tap this vast source of tumor Ags,we generated a murine IgG2a mAb,3.2G1,endowed with TCR-like binding specificity for peptide-HLA-A*0201 (HLA-A2) complex and designated this class of Ab as TCR mimics (TCRm). The 3.2G1 TCRm recognizes the GVL peptide (GVLPALPQV) from human chorionic gonadotropin beta presented by the peptide-HLA-A*0201 complex. When used in immunofluorescent staining reactions using GVL peptide-loaded T2 cells,the 3.2G1 TCRm specifically stained the cells in a peptide and Ab concentration-dependent manner. Staining intensity correlated with the extent of cell lysis by complement-dependent cytotoxicity (CDC),and a peptide concentration-dependent threshold level existed for the CDC reaction. Staining of human tumor lines demonstrated that 3.2G1 TCRm was able to recognize endogenously processed peptide and that the breast cancer cell line MDA-MB-231 highly expressed the target epitope. The 3.2G1 TCRm-mediated CDC and Ab-dependent cellular cytotoxicity of a human breast carcinoma line in vitro and inhibited in vivo tumor implantation and growth in nude mice. These results provide validation for the development of novel TCRm therapeutic reagents that specifically target and kill tumors via recognition and binding to MHC-peptide epitopes. View Publication

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however,the underlying mechanisms remain unclear. In the present study,we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly,treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus,through the Smad pathway,autocrine TGF-beta maintains uPA expression through facilitated protein secretion,thereby supporting tumor cell invasiveness,whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells. View Publication

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here,we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However,this effect is at the expense of HSC function,because continued Hh activation during regeneration represses expression of specific cell cycle regulators,leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo. View Publication

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors,which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype,loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds,which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently,the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore,Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally,we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors. View Publication

Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report,we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method,only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently,we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures,whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry,we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks,indicating that these cells contained rSRA activity. In contrast,CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally,suggesting that the lack of reconstitution was not due to homing defects. In conclusion,we show that after in vitro expansion,rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential,mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells. View Publication

The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the C" of the carboxyl-terminal CaaX motif). After farnesylation� View Publication

Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with monosomy 7 were cultured with 400 ng/ml GCSF,all samples showed significant increases in the proportion of monosomy 7 cells. In contrast,bone marrow from karyotypically normal aplastic anemia,myelodysplastic syndrome,or healthy individuals did not show an increase in monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and monosomy 7,GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA,CD34 cells showed increased expression of the GCSFR class IV mRNA isoform,which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in monosomy 7 CD34 cells,with increased phosphorylated signal transducer and activation of transcription protein,STAT1-P,and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting monosomy 7 cells. The abnormal response of monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated monosomy 7 clones expressing the class IV GCSFR,which is defective in signaling cell maturation. View Publication

Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W),which is allelic with the c-kit proto-oncogene. We show that KL,a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors,specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10,and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor,KL. View Publication

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However,the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a neuropeptide� View Publication

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However,less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved,we show that absence of MyD88,a key innate Toll like receptor signal adaptor,abrogates this resistance and facilitates inducible allograft acceptance. In our model,absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+ CD25+ regulatory T cells. Therefore,this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential,clinically relevant strategy to facilitate transplantation tolerance. View Publication

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10),which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however,there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore,the involvement of resident airway immune cells,predominantly bronchoalveolar macrophages,in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard,our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release,which occurred in a time- and dose-dependent manner. Furthermore,HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus,this work supports a model,wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo. View Publication

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor,c-Kit,and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency,both in cell-free assays and in cells,thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene,but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition,we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416,but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally,SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases. View Publication