参考文献

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation),non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study,we demonstrate that nitrogen-containing bisphosphonates (risedronate,alendronate,pamidronate,and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts,human osteoclastoma-derived osteoclasts,and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes,loss of mitochondrial membrane potential,and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed,dying osteoclasts and osteoclast-like cells using a cell-permeable,fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation,because apoptosis resulting from alendronate,clodronate,or zoledronic acid treatment was suppressed by zVAD-fmk,a broad-range caspase inhibitor,or by SB-281277,a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore,caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts,whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3,therefore,appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins,because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298,a specific inhibitor of protein geranylgeranylation,whereas FTI-277,a specific inhibitor of protein farnesylation,had no effect on apoptosis or caspase activity. View Publication

The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here,we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells,and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16,but not doxorubicin,transiently induced NF-kappaB activity in all cell lines,whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells,but rather high in Capan-1 and A818-4 cells,as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin,MG132 and Sulfasalazine),or transfection with the IkappaBalpha super-repressor,strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies. View Publication

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study,we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover,two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion,we used an in vitro liquid culture system. For this purpose,CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure,megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes,implying the presence of inhibitory factors in plasma. However,some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma,higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore,Cellgro medium is not the best alternative medium. In cultures with Stemspan medium,higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides,AF13948 and PK1M,were tested for their ability to replace Tpo. In cultures with AF13948,comparable numbers of megakaryocytes were obtained as in the presence of Tpo,but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation,but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol,it is worthwhile to test several media,because the number of megakaryocytes differs widely with the medium used. View Publication

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication

The effect of resiquimod (R-848),an immune-response modifier that is similar to imiquimod,on recurrent herpes simplex virus (HSV) was evaluated using the guinea pig model of genital herpes. Guinea pigs were intravaginally infected with HSV-2 and then were randomized on day 14 to receive nothing or 0.1 mL/kg per dose of subcutaneous resiquimod,given either daily,every other day,or weekly from days 15-35. During a 3-week course of therapy,recurrences in all 3 treated groups were reduced by textgreater80%,compared with the control group. After therapy,recurrences remained significantly (Ptextless.05) decreased in all 3 groups for the next 3 weeks. The group treated weekly developed the fewest recurrences. Significant increases in interleukin-2 levels,produced by incubation of mononuclear cells with HSV-2 antigens,but not in circulating antibody also were detected in the treated groups. Resiquimod treatment may offer significant advantages to present antiviral therapies for the control of recurrent genital herpes. View Publication

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule,truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications,as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis,where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7,2061-2070. View Publication

In Treponema denticola,a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola,and double- crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects,including highly condensed chromosomal DNA,compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover,chains of cells are formed by the cytoplasmic filament- deficient mutant,and those cells show reduced spreading in agarose,which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure,segregation,or the cell division process in Treponema. View Publication

The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10,X-vivo 15,[Bio-Whitacker,Walkersville,MD] and StemSpan [Stem Cell Technologies,Vancouver,BC,Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used,and clonogenic cells were maintained after in vitro culture in all media. In contrast,constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines,but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines. View Publication

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo,although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently,several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase,thereby preventing protein prenylation in osteoclasts. In this study,we examined the potency of a wider range of nitrogen-containing bisphosphonates,including the highly potent,heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro,to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro,and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations textgreater or = 1 nM zoledronic acid or minodronate,the order of potency (zoledronic acid approximately equal to minodronate textgreater risedronate textgreater ibandronate textgreater incadronate textgreater alendronate textgreater pamidronate) closely matching the order of antiresorptive potency. Furthermore,minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates,giving rise to less potent inhibitors of bone resorption in vivo,also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo,and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase. View Publication

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1,an inhibitor of cell cycle progression in yeast. At present,mDYRK-3 and mDYRK-2 have been cloned,and mDYRK-3 has been characterized with respect to kinase activity,expression among tissues and hematopoietic cells,and possible function during erythropoiesis. In sequence,mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a,but is 91.3% identical overall to hDYRK-3. Catalytically,mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a,mDYRK-2,and mDYRK-3 was high in testes,but unlike mDYRK1a and mDYRK 2,mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells,however,mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells,expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone),and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly,antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus,it is proposed that mDYRK-3 kinase functions as a lineage-restricted,stage-specific suppressor of red cell development. (Blood. 2001;97:901-910) View Publication