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Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers,although antitumor responses are often heterogeneous,and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug,we studied the action of imatinib (STI-571,Gleevec) against murine cells and leukemias expressing BCR-ABL,an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53,which can accompany disease progression in human CML,impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly,p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However,the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g.,the brain) is higher than for treatment of anemia. Notably,a dose-dependent risk of adverse effects has been associated with rhEPO administration,especially in high-risk groups,including polycythemia-hyperviscosity syndrome,hypertension,and vascular thrombosis. Of note,several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity,primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover,we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here,we demonstrate that rhEPO administration in the rat increases systemic blood pressure,reduces regional renal blood flow,and increases platelet counts and procoagulant activities. In contrast,carbamylated rhEPO,a heteromeric receptor-specific ligand that is fully tissue protective,increases renal blood flow,promotes sodium excretion,reduces injury-induced elevation in procoagulant activity,and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands,which appear to elicit fewer adverse effects,may be especially useful in clinical settings for tissue protection. View Publication

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However,it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3,a small molecule inhibitor of the p53/MDM2 interaction,is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells,but not on normal CD19(+) B lymphocytes,peripheral-blood mononuclear cells,or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined,only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway,nutlin-3 up-regulated the steady-state mRNA levels of PCNA,CDKN1A/p21,GDF15,TNFRSF10B/TRAIL-R2,TP53I3/PIG3,and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover,nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL. View Publication

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates,its activity in combination with other antiretrovirals,and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L,T215Y/F,plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V,Y115F,and M184V plus one other NAM) or stavudine (V75T/M,M41L,T215F/Y,and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold,respectively (these changes gave values comparable to or less than the corresponding values for zidovudine,lamivudine,abacavir,and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades,including A/G,B,C,D,A(E),D/F,F,and H. SPD754 showed additive effects in combination with other NRTIs,tenofovir,nevirapine,or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro,SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity. View Publication

Multiple myeloma is a highly radiosensitive skeletal malignancy,but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model,we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic,syngeneic 5TGM1 myeloma model,the median survivals of mice treated with saline,2 doses of PS-341 (0.5 mg/kg),or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21,22,and 28 days,respectively. In contrast,mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg),1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P textless .001). In addition to prolonged survival,this treatment combination yielded reduced clonogenicity of bone marrow-resident 5TGM1 cells,reduced serum myeloma-associated paraprotein levels,and better preservation of bone mineral density. Myelosuppression,determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors,did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent,selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma. View Publication

To investigate retinal involvement in chronic Chagas' disease,we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency,under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors,all related to the same subfamily of G-protein-coupled receptors. View Publication

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic,and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However,we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast,immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy. View Publication

BACKGROUND: The dose-limiting toxicity of doxorubicin on hematopoietic stem cells reduces the maximum benefit from this powerful drug. Melatonin may play a role in reducing this toxicity. MATERIALS AND METHODS: Melatonin at 10 microM was used while challenging human peripheral blood stem cells (PBSC) with doxorubicin (0.6 microM and 1 microM),and colony formation was used to evaluate the protective effect of melatonin. RESULTS: Melatonin was protective for the myeloid and erythroid series when given during or 1 hour after,but not before,doxorubicin,as measured by colony assay. This protection was independent from its antioxidant function as measured by 2',7'-dichlodihydro-fluorescein diacetate and was selective for PBSC when compared to the MCF-7 cancer cell line. CONCLUSION: The results suggest the importance of the time sequence for melatonin administration to exert its protective effect in relation to doxorubicin treatment,as well as its protective effect on both erythroid and myeloid elements independent from its antioxidant function. View Publication

For antibody-based therapy of cancer,monoclonal antibodies (mAbs) of human origin are superior to mouse,mouse/human chimeric or humanized mAbs,because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study,human mAbs were prepared against a pancarcinoma antigen,MK-1 (Ep-CAM),using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice,immunized with recombinant MK-1,were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed,two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants,their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity,respectively,against MK-1-expressing cells in vitro,suggesting that these fully human mAbs produced against MK-1 and their V-region genes,which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer. View Publication

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context,little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional,biochemical,and microarray analyses. When deprived of natural ligands,GM-CSF,interleukin-3,or erythropoietin,AP20187 prevented apoptosis of transduced TF1 cells,induced dose-dependent proliferation,and supported long-term growth. In addition,AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably,mRNAs affiliated with heat stress,including Hsp70 and Hsp105,were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence,in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor,or KDR,signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes. View Publication

HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function,manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. View Publication