CD34-positive Hematopoietic Cells Research Articles

AML-399: A Pan-BET Degrader Shows Broad Pre-Clinical Activity in a Large Acute Myeloid Leukemia Cohort

Bromodomain and Extra Terminal (BET) proteins are ubiquitously expressed epigenetic “readers” that play a major role in regulating expression of oncogenic genes such as c-Myc. BET protein family are crucial for acute myeloid leukemia (AML) maintenance, and expression of BRD4 is a poor prognostic indicator in AML. We have developed novel and a highly potent proteolysis-targeting, chimera (PROTAC)-based pan-BET degrader, QCA570, that depletes BET. This BET-degrader is derived from a new class of inhibitor, HYD276. We tested QCA570 in a panel of 104 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSCs) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors and drugs for 72 hrs, and cell death and apoptosis induction was measured by flow cytometry for DAPI-Annexin V binding. Primary AML cells were obtained from newly diagnosed AML (N=89) or relapse/refractory AML (N=15). The median IC50 for BET-degrader was 120 pM, and only 13 samples had an IC50 >1 nM. In contrast, the median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 μM. An IC50 of greater than 10 μM HYD276 was considered as resistant to the BET inhibitor. BET inhibitor-resistant LSCs were found to be sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBP-alpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype, and 55% had abnormal karyotype. The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4-hr exposure of LSCs to the degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in an apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of this new class of drugs in patient-derived xenografts is ongoing. In conclusion, QCA570 represents a highly potent and efficacious pan-BET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias. Bromodomain and Extra Terminal (BET) proteins are ubiquitously expressed epigenetic “readers” that play a major role in regulating expression of oncogenic genes such as c-Myc. BET protein family are crucial for acute myeloid leukemia (AML) maintenance, and expression of BRD4 is a poor prognostic indicator in AML. We have developed novel and a highly potent proteolysis-targeting, chimera (PROTAC)-based pan-BET degrader, QCA570, that depletes BET. This BET-degrader is derived from a new class of inhibitor, HYD276. We tested QCA570 in a panel of 104 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSCs) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors and drugs for 72 hrs, and cell death and apoptosis induction was measured by flow cytometry for DAPI-Annexin V binding. Primary AML cells were obtained from newly diagnosed AML (N=89) or relapse/refractory AML (N=15). The median IC50 for BET-degrader was 120 pM, and only 13 samples had an IC50 >1 nM. In contrast, the median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 μM. An IC50 of greater than 10 μM HYD276 was considered as resistant to the BET inhibitor. BET inhibitor-resistant LSCs were found to be sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBP-alpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype, and 55% had abnormal karyotype. The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4-hr exposure of LSCs to the degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in an apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of this new class of drugs in patient-derived xenografts is ongoing. In conclusion, QCA570 represents a highly potent and efficacious pan-BET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias.

AML-397: Preclinical Efficacy of a PROTAC-based MDM2 Degrader in AML models

Mouse double minute 2 homolog (MDM2) is a E3 ubiquitin ligase that promotes p53 tumor suppressor degradation. MDM2 has emerged as a therapeutic target in the treatment of TP53 wild-type expressing acute myeloid leukemia (AML) where MDM2 is overexpressed. Several MDM2 inhibitors are currently in clinical trials. However, p53 stabilization upregulates MDM2, which limits the clinical efficacy of the inhibitors. Proteolysis Targeting Chimeric (PROTAC) molecules recruit targeted proteins for degradation. We developed a MDM2 degrader, MI-265, that binds to MDM2 and targets it for degradation. We tested MI-265 in a panel of 101 primary AML samples in ex vivo cultures. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. After treatment with drugs for 72 hrs, cell death and apoptosis induction were measured by flow-cytometry for DAPI-Annexin V binding. The AML cells were obtained from newly diagnosed AML (N=86) or relapse/refractory AML (N=15). The median IC50 for MI-265 was 120 pM. In contrast, median IC50 for the inhibitor MI-1061 was 20,000-fold higher at 2.3 μM. Sensitivity and resistance to MDM2-degrader and inhibitor were similar in most of the samples suggesting that the mechanism of action of both drugs are also similar. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype and 55% had abnormal karyotype. The MDM2 degrader-resistant LSCs (IC50 greater than 1 μM) were analyzed for TP53 mutation status and 8 of 14 were found to harbor mutations in TP53. The degradation of MDM2 and overexpression of p53 was confirmed by immunoblotting after a 4-hr exposure of LSCs to degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of efficacy in patient-derived xenografts is ongoing. In conclusion, MI-265 represents a highly potent and efficacious MDM2 degrader, and is currently undergoing extensive preclinical evaluation for the treatment of acute leukemias.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly

AbstractApoptosis resistance contributes to poor outcome in pediatric acute lymphoblastic leukemia (ALL). Here, we identify a novel synergistic combination of Smac mimetic BV6 and glucocorticoids (GCs) (ie, dexamethasone, prednisolone) to trigger apoptosis in ALL cells. BV6 and GCs similarly cooperate to induce apoptosis in patient-derived leukemia samples, underlining the clinical relevance. Importantly, BV6/dexamethasone cotreatment is significantly more effective than monotherapy to delay leukemia growth in a patient-derived xenograft model of pediatric ALL without causing additional side effects. In contrast, BV6 does not increase cytotoxicity of dexamethasone against nonmalignant peripheral blood lymphocytes, mesenchymal stromal cells, and CD34-positive hematopoietic cells. We identify a novel mechanism by showing that BV6 and dexamethasone cooperate to deplete cIAP1, cIAP2, and XIAP, thereby promoting assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing complex. This complex is critical and is required for BV6/dexamethasone-induced cell death, because RIP1 knockdown reduces caspase activation, reactive oxygen species production, and cell death. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, because blocking antibodies for TNFα, tumor necrosis factor-related apoptosis-inducing ligand, or CD95 ligand or knockdown of death receptors fail to rescue BV6/dexamethasone-induced cell death. This is the first report showing that BV6 sensitizes for GC-triggered cell death by promoting ripoptosome formation with important implications for apoptosis-targeted therapies of ALL.

Rb and nucleolin antagonize in controlling human CD34 gene expression

Retinoblastoma protein (Rb) controls cell proliferation, differentiation, survival and gene expression and it has a central role in the signaling network that provides a cell cycle checkpoint in the G1 phase of the cell cycle. Studies in mice have shown that Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment and it acts as a critical regulator of hematopoietic stem and progenitor cells under stress. In human hematopoiesis, the CD34 protein is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution, and CD34 has a role in the differentiation of hematopoietic cells. Here we find that, in CD34-positive hematopoietic cells, Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin to provide a mechanism that links expression of endogenous CD34 to cell cycle progression. Our study suggests a direct involvement of Rb in the transcriptional program of human CD34-positive hematopoietic stem/progenitor cells, thus providing further insights into the molecular network relevant to the features of these cells.

Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

Nucleolin Regulates Gene Expression in CD34-positive Hematopoietic Cells

CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution. Nucleolin is an abundant multifunctional phosphoprotein of growing eukaryotic cells, involved in regulation of gene transcription, chromatin remodeling, and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we show that, in human CD34-positive hematopoietic cells, nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative peripheral blood mononuclear cells (PBMCs). Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained.

Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis

Extravasation of leukocytes is a crucial process in the immunological defense. In response to a local concentration of chemokines, circulating leukocytes adhere to and migrate across the vascular endothelium toward the inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role in the regulation of leukocyte adhesion, polarization, and chemotaxis. We investigated the role of a guanine nucleotide exchange protein for Rap1 directly activated by cAMP (Epac1) in adhesion and chemotaxis in a promonocytic cell line and in primary monocytes. We found that Epac1 is expressed in primary leukocytes, platelets, CD34-positive hematopoietic cells, and the leukemic cell lines U937 and HL60. Epac activation with an Epac-specific cAMP analog induced Rap1 activation, beta1-integrin-dependent cell adhesion, and cell polarization. In addition, activated Epac1 enhanced chemotaxis of U937 cells and primary monocytes. Similar to activation of Epac1, stimulation of cells with serotonin to induce cAMP production resulted in Rap1 activation, increased cell adhesion and polarization, and enhanced chemotaxis. The effects of serotonin on U937 cell adhesion were dependent on cAMP production but could not be blocked by a protein kinase A inhibitor, implicating Epac in the regulation of serotonin-induced adhesion. In summary, our work reveals the existence of previously unrecognized cAMP-dependent signaling in leukocytes regulating cell adhesion and chemotaxis through the activation of Epac1.

Development of a New Experimental Animal Model of Human Megakaryopoiesis Using Immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) Mice.

[Background] We developed novel small molecules with thrombopoietin (TPO) mimetic activities in vitro. These small molecules induced colony formation of megakaryocytes from human bone marrow cells but not murine and cynomolgus monkey cells, indicating strict species specificity. However, the lack of experimental animal models for human megakaryopoiesis hampered evaluation of whether these molecules can increase the number of human platelets in vivo. To solve this problem, we developed a new experimental animal model of human megakaryopoiesis using immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice.[Aims] We developed a new experimental animal model of human megakaryopoiesis using NOG mice to evaluate the in vivo efficacies of the novel human-specific TPO mimetics. We also compared the potentials of cord blood (CB), bone marrow (BM), and peripheral blood (PB)-derived CD34 positive cells at maintaining human megakaryopoiesis in NOG mice.[Methods] After 2.4-Gy X-ray irradiation, 1x105 CD34-positive hematopoietic cells from human CB, BM and PB were intravenously injected into NOG mice. The engraftment of human megakaryocytes and platelets in NOG mice was analyzed by flow cytometry and immunohistochemistry with human-specific antibodies for up to 6 months.[Results] Four weeks after transplantation, we found that around 1% of platelets in these mice were positive for human CD41 antigen (fibrinogen receptor) (n=8). We also confirmed 30–40% of megakaryocytes in the murine bone marrow were positive for human-specific megakaryocyte markers by immunohistochemistry. The percentage of human platelets in the murine blood was maintained at around 2% for 6 months after transplantation with human CD34-positive CB cells. In contrast, the engraftment of human platelets was transient and almost disappeared after 3 months, when CD34-positive cells from human BM and PB were injected into NOG mice. Interestingly, we found around 20 and 40% of murine bone marrow cells were positive for human CD45 three months after transplantation with PB- and BM-derived CD34-positive cells, respectively (n=7). These results indicated that CB cells have higher potencies to maintain human megakaryopoiesis than BM and PB cells in NOG mice. As the percentage of human platelets was much lower than that of human megakaryocytes in NOG mice transplanted with human CB cells, we speculated that the clearance of human platelets, which are larger than murine platelets, might be high or that the murine bone marrow microenvironment lacks factors related to platelet release from human megakaryocytes. We also analyzed the function of human platelets in NOG mice. ADP induced the activation of alpha IIb beta IIIa (fibrinogen receptor) and the expression of CD62P on human platelets in NOG mice in vitro, suggesting that human platelets produced from human CB-derived CD34 positive cells in NOG mice, are functional.[Conclusions] We succeeded to develop a new experimental animal model of human megakaryopoiesis using NOG mice. CB was the most suitable donor for maintaining human matured megakaryocytes and functional platelets in NOG mice. Currently, we are using this model to evaluate in vivo efficacies of novel human-specific TPO mimetics.

Demethylation Profiling of CD34-Positive Hematopoietic Cells in Patients with Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a genetic and epigenetic disease of the hematopoietic stem cell. Aberrant CpG islands methylation in the contex of the promoter of multiple genes plays a pivotal role in the pathogenesis of MDS and leads to silencing of tumor suppressor genes, including cell-cycle inhibitors, inducers of apoptosis, DNA repair genes, transcription factors, cell adhesion mediators, hormonal receptors and detoxifiers. Demethylating agents, such as decitabine and azacitidine, are able to revert epigenetic silencing induced by hypermethylation and are currently used to treat all subtypes of MDS. Some of the target genes of demethylating drugs have been well studied and correlated to clinical response of patients, such as p15INK4B, but most of them remain to be identified and characterized. We isolated CD34+ cells from two patients with previously untreated MDS, a 70 year old female, with a diagnosis of Refractory Anemia with Excess Blasts (RAEB) and a complex karyotype including deletion of 5q11–q34 and trisomy 8, and a 59 years old male, with a diagnosis of RAEB in transformation, according to FAB and a normal karyotype. CD34+ cells were isolated from bone marrow samples by immunomagnetic beads, with a yield of about 2 x 106 cells per patients. Purity of the CD34+ cell fraction, evaluated by flow cytometry, was 58% and 86%, respectively. Cells were cultured in 24-well plates in IMDM medium with L-Glutamine, antibiotics, 30% of inactivated Foetal Bovine Serum and 10 ng/ml each of IL-3, Stem Cell Factor (SCF), Thrombopoietin and FLT3-ligand. After 24 hours, decitabine was added to the culture medium to a final concentration of 1 m M. A corresponding amount of acetic acid was added to different wells for the mock treatment control. Each experiment was conducted in triplicate. Cells were collected after 72 hours of treatment and RNA was extracted by the Qiagen RNeasy Kit, processed by two-cycle cDNA synthesis kit (Invitrogen), in vitro transcripted to cRNA and hybridized on Affymetrix HG-U133A chips. Five chips were used for each patient: three for treated cells and two for mock -treated cells. Microarray data were normalized and analysed by GeneSpring software version 7.2 and the ANOVA Welch's test was applied. We selected genes with a p value less than 0.01 and a fold change higher than 2. Using these conditions, 60 genes were upregulated by decitabine in both patients. Some of the most interesting genes were GATA binding protein 2 (GATA2), cyclin-dependent kinase inhibitor 1A (CDKN1A, p21), cyclin A1 (CCNA1), decay accelerating factor for complement (CD55, DAF), immediate early response 3 (IER3), nuclear factor interleukin 3 regulated (NFIL3) and chemokine (C-X-C motif) receptor 4 (CXCR4).Interestingly, the patient with a normal karyotype showed a higher percentage of up-regulated genes after decitabine treatment compared to the patient with the 5q11-q34 deletion and a trisomy 8. This suggests that epigenetic changes in gene expression may have higher impact when the karyotype is normal. Functional significance of these data remains to be elucidated. Expression and methylation status of these genes will be investigated in a larger group of MDS patients. This approach aims to characterize new genes, as methylation targets in MDS and possible markers of disease, and to identify patients responding to demethylating agents.

CD34 Class III positive cells are present in atherosclerotic plaques of the rabbit model of atherosclerosis

CD34 is a positive marker for haematopoietic stem cells and endothelial cells. Recent evidence suggests that haematopoietic progenitor cells are involved in atherogenesis. CD34-positive haematopoietic progenitor cells have never been described in rabbit atherosclerotic tissues. The aim of this study is to identify CD34-positive haematopoietic progenitor cells in rabbit atherosclerotic tissues, and to compare this with macrophage (RAM-11), alpha smooth muscle cell actin and fibroblast (prolyl-4-hydroxylase) immunoreactive cells. Sixteen Male New Zealand White rabbits were divided into two groups: Group 1, control diet (Con); group 2, 0.5% cholesterol diet, and killed after 12 weeks. Immunohistochemistry was used to detect CD34 haematopoietic progenitor cells. CD34-positive haematopoietic progenitor cells were identified both within and overlying atherosclerotic plaques. As well, these haematopoietic progenitor cells also stained for RAM-11, CD45, prolyl-4 hydroxylase and alpha smooth muscle cell actin. These findings suggest that in the rabbit model of atherosclerosis, the previously identified macrophages, smooth muscle cells and fibroblasts within and overlying atherosclerotic plaques might be of haematopoietic origin.

Proteinated Elemental Selenium Potentiates Anti-Tumor Effect of Ionizing Radiation and Chemotherapeutic Agents but Is Only Minimally Affected by Drug Resistance Mechanisms.

Elemental selenium generated by the photobleaching of selone photosensitizers combined with serum albumin and lipoproteins to generate conjugates that were highly cytotoxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic cells. Selenium-protein conjugates were internalized by an endocytotic mechanism and localized in lysosomes. Internalized conjugates caused an extremely rapid and extensive (up to 80% in 1 hour) depletion of intracellular glutathione (GSH), the oxidation of 2′,7′-dichlorofluorescin, a loss of plasma membrane asymmetry, a breakdown of mitochondrial potential, and the activation of several caspases. Additive or synergistic anti-tumor effects were obtained when Se-protein conjugates were used in combination with ionizing radiation, melphalan, amifostine, 4-hydroperoxycyclophosphamide, edelfosine, buthionine sulfoximine, or moderate doses of arsenic (III) oxide. For example, pretreatment with a nontoxic dose of amifostine enhanced the depletion of L1210 leukemia cells from 7 log to 12 log (depletions in excess of 6 log estimated from linear extrapolations of survival curves) while the yield of normal CD34-positive cells remained at 100%. Antagonistic interactions were obtained with simultaneously applied cisplatin or very high doses of arsenic (III) oxide. The antagonistic interaction with cisplatin probably reflected a competition of cisplatin and selenium for the same binding site at or near cysteine-34 of albumin. Melphalan-resistant L1210/L-PAM1 leukemia cells (elevated intracellular GSH) were more sensitive to Se-protein conjugates than wild-type cells because of an unexpected increase in conjugate uptake by mutant cells. Cisplatin-resistant H69/CDDP cells (elevated GSH, metallothioneine, glutathione-S-transferase-pi) were slightly more sensitive than wild-type cells. Cisplatin-resistant PC14/CDDP cells (reduced drug uptake) and adriamycin-resistant HL-60/ADR cells (MRP-mediated efflux) were slightly less sensitive than the corresponding wild-type cells. In the latter cases, the reduced sensitivity of mutant tumor cells correlated with reduced conjugate uptake and/or higher intracellular GSH levels. Taken together, the above results suggest that compounds modeled after our Se-protein conjugates may prove useful for the systemic therapy of leukemia and selected solid tumors. Supported by CA77387, MACC Fund, Children's Hospital Foundation, and Wisconsin Breast Cancer Showhouse for a Cure.

Genome size and structure determine efficiency of postinternalization steps and gene transfer of capsid-modified adenovirus vectors in a cell-type-specific manner.

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.

Molecular cloning of feline CD34

In humans, baboons, dogs and mice CD34 is a cell surface molecule that is expressed on primitive hematopoietic cells and in all these species CD34 positive cells can be used to effect long-term haematopoietic reconstitution. CD34 positive haematopoietic cells therefore provide a convenient and relatively small cell population to target when attempting gene therapy via the haematopoietic system. In order to develop the mucopolysaccharidosis type VI (MPS VI) cat as a model for haematopoietic cell-mediated gene therapy we have isolated the feline CD34 gene as a first step in the generation of antibodies for purification of feline CD34 positive cells. The coding sequence for feline CD34 was isolated from brain cDNA using the polymerase chain reaction (PCR) with oligonucleotides designed to conserved regions of known CD34 gene sequences as primers. Sequence analysis of PCR products revealed the complete amino acid sequence of feline CD34 and allowed analysis of sequence conservation with CD34 from other species. Northern blot analysis showed a 2.6 kb CD34 transcript was present in feline brain, spleen, heart, testis and thymus, and to a lesser extent, in liver. A full-length cDNA clone of the feline CD34 coding sequence was assembled and expressed in CHO-K1 cells. The isolation and expression of the feline CD34 cDNA should facilitate the production of antibodies suitable for the purification of CD34 positive cells.

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2′,7′-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

Induction of apoptotic change of hematopoietic cells by cloned stromal cells from mds patient via direct cellular interactions

We previously reported on the heterogeneity of bone marrow stromal cell function in supporting hematopoietic cell proliferation and differentiation in vitro among refractory anemia (RA) of myelodysplastic syndrome (MDS) patients. Interestingly, stromal cells from same MDS RA patients induced an apoptotic change in CD34-positive hematopoietic cells (CD34+). To clarify the mechanism of this action of stromal cells, a cloned stromal cell line (LS801) from MDS patient was established. In a coculture system with LS801, proliferation and differentiation of CD34+ cells was inhibited. Assay for the expression of 7A6 antigen on CD34+ cells, for monitoring the cells undergoing apoptosis, showed that LS801 induced an apoptotic change in CD34+ cells. When CD34+ cells were cocultured but kept separate fom LS801 by a 0.45-mm millipore membrane to prevent their attachment, the action of LS801 for inducing apoptosis of CD34+ cells was inhibited. The expression of CD95 on CD34+ cells was induce by LS801, however, no production of fas ligand, TNF-α or IFN-γ in LS801 was observed. In light of these observations, direct contact between LS801 and CD34+ cells might be essential for maximum induction of CD34+ cells undergoing apoptosis.

CD34/QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes.

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p<0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p<0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.