技术资料

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication

DNA-dependent protein kinase (DNA-PK),which is involved in DNA double-stranded break repair and V(D)J recombination,comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit,DNA-PKcs. Here,we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays,however,indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly,DNA-PKcs is most similar to PI kinase family members involved in cell cycle control,DNA repair,and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p,Tor2p,and FRAP,S. pombe rad3,and the product of the ataxia telangiectasia gene,mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action. View Publication

Although natural killer (NK) cells are well known for their ability to kill tumors,few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here,we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1,ULBP2,and ULBP3. In line with the NK receptor ligand expression profiles,antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. View Publication

Cancer stem-like cells represent poorly differentiated multipotent tumor-propagating cells that contribute disproportionately to therapeutic resistance and tumor recurrence. Transcriptional mechanisms that control the phenotypic conversion of tumor cells lacking tumor-propagating potential to tumor-propagating stem-like cells remain obscure. Here we show that the reprogramming transcription factors Oct4 and Sox2 induce glioblastoma cells to become stem-like and tumor-propagating via a mechanism involving direct DNA methyl transferase (DNMT) promoter transactivation,resulting in global DNA methylation- and DNMT-dependent downregulation of multiple microRNAs (miRNAs). We show that one such downregulated miRNA,miRNA-148a,inhibits glioblastoma cell stem-like properties and tumor-propagating potential. This study identifies a novel and targetable molecular circuit by which glioma cell stemness and tumor-propagating capacity are regulated. View Publication

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy,many of them subsequently relapse,and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A),most frequently at arginine 882 (DNMT3A(R882)),have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A(R882) AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy,suggesting that DNMT3A(R882) cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion,cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3(ITD)) and the nucleophosmin gene (Npm1(c)) to induce AML in vivo,and promoted resistance to anthracycline chemotherapy. In patients with AML,the presence of DNMT3A(R882) mutations predicts minimal residual disease,underscoring their role in AML chemoresistance. DNMT3A(R882) cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment,which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress,which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A(R882) mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy. View Publication

During the first month of pregnancy,the brain and spinal cord are formed through a process called neurulation. However,this process can be altered by low serum levels of folic acid,environmental factors,or genetic predispositions. In 2018,a surveillance study in Botswana,a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs,found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result,the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs,we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing,Optical Coherence Tomography (OCT),Optical Coherence Elastography (OCE),and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast,reverberant OCE measurements indicate decreased organoid volumes and internal stiffness. View Publication

Donor cell neoplasms are rare complications of treatment regimens that involve stem cell transplantation for hematological malignancies,myelodysplastic processes,or certain genetic or metabolic disorders. We report a case of donor cell leukemia in a pediatric patient with a history of acute myeloid leukemia that manifested as recurrent AML FAB type M5 fourteen months after umbilical cord blood transplantation. Although there was some immunophenotypic drift from the patient's original AML and their posttransplant presentation,the initial pathological impression was of recurrent disease. Bone marrow engraftment analysis by multiplex PCR of short tandem repeat markers performed on the patient's diagnostic specimen showed complete engraftment by donor cells,with a loss of heterozygosity in the donor alleles on chromosome 7. This led to the reinterpretation of this patient's disease as donor-derived leukemia. This interpretation was supported by a routine karyotype and fluorescence in situ hybridization analysis showing loss of chromosome 7 and a male (donor) chromosome complement in this female patient. Also noted was a loss of the patient's presenting chromosomal abnormality,t(11;19)(q23;p13). This case highlights the need for close coordination between all aspects of clinical testing for the transplant patient,including molecular engraftment studies,when distinguishing the very common complication of recurrent disease from the exceedingly rare complication of donor cell leukemia. View Publication

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation,DAF has been thought to primarily regulate antibody-mediated allograft injury,which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses,we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response,in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression,by controlling local complement activation on interacting T cell APC partners,regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation. View Publication

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients,respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (textgreater or = 98%) for CD73,CD105,CD106,CD29,CD13,CD44 and negative (textless or = 1%) for CD34,CD45,CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients,respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes,a mature subpopulation of peripheral blood monocytes,are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy,continued influx of CD14+CD16+ monocytes,both infected and uninfected,contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain,CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study,we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R,D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist,SKF38393,increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion,live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay,dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition,adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions. View Publication

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However,the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease,DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo,suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling,biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells,complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally,scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival,function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease. View Publication