Development of Leukemia after HOXB4 Gene Transfer in the Canine Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 204-204 ◽  
Author(s):  
Xiao-Bing Zhang ◽  
Brian C. Beard ◽  
Grant Trobridge ◽  
Robert Hackman ◽  
Eileen Bryant ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Canine Model ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Dominant Band ◽  
Blast Cells ◽  
Exon 4

Abstract HOXB4 is considered to be the only HOX gene that promotes self-renewal of hematopoietic stem cells without causing leukemia in mouse models. We investigated whether HOXB4 overexpression has similar effects in a clinically relevant canine model. A competitive repopulation assay was performed in three dogs in which CD34+ cells were transduced with MSCV-based gammaretroviral vectors expressing HOXB4GFP or control YFP. We observed up to 4-fold higher marking levels in granulocytes for the HOXB4GFP arm relative to the control 1 month after transplantation. The marking levels eventually decreased in all three animals and two dogs (G374, G450) have now been followed for more than 18 months. In G374, the marking levels for both arms stabilized at ~2% after 2 months post-transplantation. Between 14 and 20 months post-transplantation, the HOXB4GFP marking steadily increased to >95%, while YFP marking decreased to 0.1%. G374 was euthanized 21 months after transplantation due to declining health. Flow cytometry analysis showed that ~50% of BM cells expressed the monocyte marker CD14 and ~8% expressed the granulocyte marker DM5, all of which also expressed HOXB4GFP. CD3 and CD21 were expressed in 2% and 1% of cells, respectively, but these cells did not express HOXB4GFP. Bone marrow necropsy demonstrated significantly increased numbers of blast cells, consistent with a myelomonocytic leukemia. Southern blot analyses of G374 BM and PB samples identified 2 bands with the same intensity, suggesting a single dominant clone with 2 integration sites. LAM-PCR analysis identified two vector proviruses integrated ~100 kb upstream of c-myb, and into intron 3 of PRDM16. Western blot analysis confirmed expression of HOXB4 in cultured G374 BM cells but the levels of c-myb in these cells were not different from control HOXB4-transduced BM cells as determined by RT-PCR. The expression of PRDM16 exons 1–3 was not detected in cells from dog G374 or in control cells, however, PRDM16 exon 4 was expressed in G374 cells but not in control cells. RT-PCR using primers located in the MSCV LTR and in PRDM16 exon 4 identified a unique band and sequencing of this product showed that the 5′ LTR was spliced with PRDM16 exon 4 creating a short PRDM16 isoform which has been observed in human leukemias. These data suggest that HOXB4 overexpression in collaboration with integration-induced activation of PRDM16 led to the leukemia. Southern blot and SYBR green Q-PCR showed that the leukemic clone contributed to ~20% hematopoiesis in BM 6 months after transplantation, and gradually decreased to ~2% before final expansion of the clone, suggesting accumulation of other mutation(s) were required for overt leukemia. Karyotype analysis of BM cells has not shown any major abnormalities but we are currently performing analyses to search for minor abnormalities such as gene duplications and deletions. Recently, HOXB4GFP marking in dog G450 PB and BM has increased to 20% and 80%, respectively, while YFP marking has decreased to ~1%. Southern blot analysis has identified a single dominant band and a BM biopsy showed substantially increased blast cells. Of note, we have not observed leukemia in >30 dogs followed long term that received transduced cells without HOXB4. In summary, HOXB4 overexpression together with insertional mutagenesis by virus integration has induced leukemia in the canine model, demonstrating the utility of this model to study the safety of gene therapy.

Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2389-2389
Author(s):  
Lev Silberstein ◽  
Masatake Osawa ◽  
Charles Lin ◽  
Peter Kharchenko ◽  
Cristina Lo Celso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Chi Squared ◽  
The Individual ◽  
Osteolineage Cells ◽  
Angiopoietin 1

Abstract Abstract 2389 Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal. To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs. Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification. As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test). Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures: Scadden: Fate Therapeutics: Equity Ownership.

Recombinant scAAV2 Vector-Mediated Ex Vivo Transduction of Primary Human Hematopoietic Stem Cells from a β-Thalassemia Patient and Human β-Globin Gene Expression in a Murine Xenograft Model in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5951-5951
Author(s):  
Liu-Jiang Song ◽  
Xin-Hua Zhang ◽  
Jun Zhu ◽  
Jue-Lian Wu ◽  
Xiao-Ling Yin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Globin Gene Expression

Abstract Background: The most severe form of β-thalassemia, β°-thalassemia major, is characterized by the complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors have been used successfully to achieve clinical efficacy in one patient, although clonal expansion of a myeloid cell population also occurred in this patient which was associated with the activation of a cellular proto-oncogene, HMGA2. We reasoned that recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), might offer a safer alternative. We have previously documented that although the conventional single-stranded (ss) AAV2 vectors mediated β-globin gene transfer and expression in primary human fetal liver cells and in human HSCs from patients with β-thalassemia patients in vitro, the level of transgene expression was sub-optimal. In the present study, we investigated whether double-stranded self-complementary (sc) AAV2 vectors could overcome this limitation. Methods: Human HSCs, obtained from a β-thalassemia homozygous patient, were mock-transduced or transduced with recombinant scAAV2-β-globin vectors at 5×104 vgs/cell, followed by i.v. injection into sub-lethally irradiated NOD/SCID mice (2.65 cGy total body irradiation), which were also pre-treated with 200 µg purified anti-IL2RB/CD122 monoclonal antibody. Recipient mice were sacrificed 12 weeks post-transplantation. Bone marrow cells from recipient mice were analyzed by BFU-E assays. Human β-globin gene expression in human erythroid progenitor cells from transplanted mice was evaluated by RT-PCR. Results: Pre-treatment of NOD/SCID mice with anti-CD122 antibody improves engraftment of human HSCs in bone marrow of receipt mice. Human β-actin (538-bp) and β-globin (272-bp) transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that recombinant scAAV2-β-globin–transduced HSCs from a patient with β-thalassemia were successfully transduced and transplanted in these mice and that human β-globin gene was transcriptionally active 12 weeks post-transplantation. Conclusion: Our results indicate that human HSCs from β-thalassemia patients can be efficiently transduced by recombinant scAAV2-β-globin vectors followed by expression of normal human β-globin gene. These studies provide the proof-of-concept that scAAV2 vector-mediated gene transfer into human HSCs might be a potentially safer alternative approach for gene therapy of β-thalassemia. Disclosures No relevant conflicts of interest to declare.

Modulation of endocardial natriuretic peptide receptors in right ventricular hypertrophy

1999 ◽  
Vol 277 (6) ◽  
pp. H2280-H2289 ◽  
Author(s):  
Sung Zoo Kim ◽  
Kyung Woo Cho ◽  
Suhn Hee Kim
Keyword(s):  
Pulmonary Hypertension ◽  
Southern Blot ◽  
Natriuretic Peptide ◽  
Blot Analysis ◽  
Binding Sites ◽  
Southern Blot Analysis ◽  
Ventricular Hypertrophy ◽  
Radioligand Binding ◽  
Left Ventricular ◽  
Rt Pcr

Natriuretic peptide (NP) receptors (NPRs) located at the endocardial endothelium are suggested to be involved in regulating myocardial contractility. However, the characteristics and modulation of NPRs in relation to cardiac failure are not well defined. This study examined the properties of NPRs in ventricular endocardium using quantitative receptor autoradiography, RT-PCR, Southern blot analysis, and activation of particulate guanylyl cyclase (GC) by NPs. In control rats, specific 125I-labeled rat atrial NP (rANP)(1–28) binding sites were localized in right (RV) and left ventricular (LV) endocardium. Binding affinities of125I-rANP(1–28) were remarkably higher in RV than LV endocardium. Radioligand binding at these sites was mostly inhibited by des[Gln18,Ser19,Gly20,Leu21,Gly22]ANP(4–23), a specific NP clearance receptor ligand. mRNAs for all three recognized NPRs were detected in endocardial cells by RT-PCR and confirmed by Southern blot analysis. Production of cGMP by particulate GC in endocardial cell membranes was stimulated by NPs with a rank order of potency of C-type NP(1–22) >> brain NP (BNP)(1–26) > ANP(1–28). We also examined the modulation of these NPRs during cardiac hypertrophy induced by monocrotaline (MCT). In MCT-treated rats with pulmonary hypertension, specific125I-rANP(1–28) binding to hypertrophied RV endocardium almost disappeared and cGMP production by NPs was significantly decreased. In rats with pulmonary hypertension, plasma levels of ANP and BNP were increased by fivefold compared with controls. The results indicate that there is a differential distribution of NPRs in the cardiac chambers, with the most abundant binding sites in RV endocardium, that NPR-B is the predominant GC-coupled NPR in ventricular endocardium, and that endocardial NPRs are downregulated with ventricular hypertrophy. Downregulation of NPRs may be associated with an increment of endogenous NP production caused by mechanical overload in hypertrophied ventricle.

Human CB-Derived Cells Give Rise to Insulin-Producing-Cells in Xenogeneic Hosts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3603-3603
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Noriaki Kawano ◽  
Hua Zhang ◽  
Yuan Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Chromosome ◽  
Human Insulin ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Insulin Producing Cells

Abstract (Purpose) We examine the capacity of human cord blood (CB)-derived cells to generate insulin-producing cells and other lineages of cells in pancreatic tissue in vivo. (Method) Ten millions of human CB-derived T-cell-depleted mononuclear cells were intravenously transplanted into conditioned newborn NOD/SCID/b2-microglobulinnull mice with or without chemical injury by an intraperitoneal injection of streptozotocin (STZ) at dose of 100 mg/g body weight. At 1–3 months post-transplantation, pancreatic tissues of the recipient mice were analyzed for the presence of human CB-derived cells by performing immunofluorescence study (insulin, amylase, or CD45) and FISH analysis for human chromosomes on the same specimens. RNA was isolated from pancreatic tissues of recipient mice, and RT-PCR analysis using human insulin specific primer was performed to examine human insulin at RNA level. Finally, double FISH analysis for human- and murine chromosomes was performed to get an insight into the mechanism for the generation of human CB-derived insulin-producing cells in vivo. (Results) At 1–3 months post-transplantation, human CB-derived T-cell-depleted mononuclear cells gave rise to both myeloid and lymphoid progeny (CD33+, CD19+, and CD3+ cells) in bone marrow and peripheral blood of the recipient mice. In recipient pancreatic tissues, human CB-derived cells were identified inside and outside islets. Outside pancreatic islets, the vast majority of human chromosome+ cells were CD45+ hematopoietic cells, while human chromosome+ amylase+ acinar cells were also identified. Inside islets, human chromosome+ cells accounted for 1.01 +/− 0.73 % (n=6) without STZ treatment. Among them, human CB-derived insulin-producing cells were identified at a frequency of 0.65 +/− 0.64 % (n=6) of total insulin+ cells in xenogeneic hosts. RT-PCR analysis demonstrated the presence of human insulin, whose sequence was fully identical to that of already-known human insulin cDNA. Chemical injury with STZ treatment led to the significant destruction of islet tissue and reduction of cell numbers in islets. In STZ-treated recipient mice, however, human insulin-producing cells were identified at a frequency of 0.23 +/− 0.27 % (n=4) in islets, which was lower than the mice without STZ treatment. Finally, double FISH analyses using species-specific probes demonstrated the presence of human chromosome+ murine chromosome+ insulin-producing cells and human chromosome+ murine chromosome- insulin-producing cells in recipient islets. (Conclusion) It is concluded that human CB cells contain the progenitor cells to generate the insulin-producing cells in vivo. The mechanism of CB-derived insulin-producing cells includes both fusion-dependent and independent mechanisms. Although the capacity of CB-derived cells needs to be compared with other stem cell sources such as tissue stem cells or embryonic stem cells, the present study suggests the possibility of CB cells as new source for future regenerative medicine for diabetes mellitus.

Natural History of Post-Allogeneic HSCT EBV-Reactivation: A Single Center Retrospective Survey.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3232-3232
Author(s):  
Helene M. Schoemans ◽  
Koen Theunissen ◽  
Marc Boogaerts ◽  
Johan Maertens
Keyword(s):  
Opportunistic Infections ◽  
Screening Tests ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Ebv Reactivation ◽  
Number Of Patients ◽  
Significant Difference

Abstract Epstein Barr Virus (EBV) reactivation occurs in about 50% of the allogeneic hematopoietic stem cell transplantation (HSCT) population in the first year post-transplantation. About 1-7% of these patients run the risk of developing a post-transplant lymphoproliferative disorder (PTLD). Several authors have thus advocated systematic screening by EBV real time PCR (RT-PCR) to initiate pre-emptive treatment of reactivations using Rituximab (van Esser 2002). However, the positive predictive value of EBV RT-PCR is only of 40% (van Esser 2001), implying that this algorithm overtreats a number of patients. Methods: We have retrospectively analyzed 60 consecutive allogeneic HSCT patients transplanted in our center between 1/1/2004 and 31/3/2005. Four patients were excluded because of absence of EBV follow-up (n=2) or autologous reconstitution (n=2). EBV reactivation (EBV (+)) was defined by at least two consecutive episodes of EBV RT-PCR above 1000 copies/ml of whole blood. Any other result was considered as negative (EBV (−)). Results: 1175 EBV RT-PCR samples were collected over a median follow up of 215 days (range: 21–511). The population observed was essentially adults (median age 42 years, range: 1–65) with leukemia (29 leukemia, 11 lymphomas, 16 other diseases), mixed graft types (26 matched sibling donors, 26 matched unrelated donors, 4 haploidentical donors; 77% peripheral blood stem cells; 20% CD34+ selection) and mixed conditioning (52% non-myeloablative conditioning containing ATG, and 48% full conditioning). The EBV(+) and EBV(−) cohorts were similar for all characteristics analyzed. We observed a median of 18 EBV RT-PCR per patient (range: 4–105), with a median interval between two tests of 7 days (range: 3–45). There were 30 true reactivations, 2 intermittent reactivations (non consecutive EBV titer rises above threshold), 8 isolated reactivations and 16 patients with no reactivation episode. EBV RT-PCR was first performed at a median of 6 days post HSCT (range: 0–245), and reactivation was noted at a median of 44 days post HSCT (range: 6–375). There were no significant difference in PCR follow up (first day of screening, median test interval and length of biological follow up) except for the total number of screening tests per patient, which was higher in the EBV(+) group (p= 0.01). There was only one case of biopsy-proven PTLD in the EBV(+) cohort. No patient was administered Rituximab post- HSCT. Survival curves of the two cohorts were similar regardless of EBV reactivation (log-rank, p= 0.201). Discussion: The incidence of EBV reactivation (n= 30; 54%) and of PTLD (n=1;1.7%) were standard compared to previous studies, resulting in a standard specificity of 47% for EBV screening. However, within our limited group of patients, we could not show any significant differences in mortality between the EBV(+) and EBV (−) cohorts. Therefore, absence of preventive treatment for EBV reactivation did not result in an increase in mortality in our EBV reactivating cohort. This suggests that systematic prophylactic use of Rituximab may not affect overall mortality, whilst potentially increasing the risk of other opportunistic infections. Conclusion: Further prospective studies are needed to better define the patients at risk for developing EBV-related PTLD, within the EBV reactivating allogeneic transplant patients group, before prophylactic treatment of reactivation becomes a routine procedure.

Downregulation of mTOR and P70S6Kβ2 in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL) Is Correlated with a Poor Prognosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2508-2508 ◽  
Author(s):  
Paola Bonaccorso ◽  
Manuela Tumino ◽  
Angela D'Ambra ◽  
Elena Mirabile ◽  
Martina Barchitta ◽  
...  
Keyword(s):  
High Risk ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Treatment Plan ◽  
Lymphoblastic Leukemia ◽  
Prognostic Impact ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Phosphorylated Akt

Abstract Abstract 2508 Background. T-ALL accounts for about 15% of pediatric ALL and more than 20% of children experience a recurrent disease which has a dismal prognosis. Characterization of molecular alterations with prognostic impact may be useful for an early identification of patients at high risk of failure in whom more intensive and/or better tailored treatments, including hematopoietic stem cell transplantation (HSCT) may be considered. PTEN/AKT pathway has been shown to be involved in children with T-ALL (Gutierrez A et al 2009), in glucocorticoid resistance (Beesley A et al 2009) and in NOTCH-1 mutated T-ALL resistant to gamma secretase inhibitors (Palomero T et al. 2009). In the attempt to better characterize the role of the PTEN/AKT/mTOR pathway in pediatric T-ALL, we have evaluated the expression of each protein in this pathway's cascade and investigated its association with outcome. Materials and Methods. We enrolled in our study 23 children with T-ALL consecutively diagnosed at our Center in Catania from 1997 to 2009. They were treated by three consecutive AIEOP protocols. We analyzed the mRNA expression of PTEN, using RT-PCR. We evaluate by western blot analysis, the expression of the following proteins: in total (AKT; GSK3β; CK2α; CK2β; PTEN; PDK1; P70S6Kβ2; mTOR; S6K) and phosphorylated [AKT(S473) (T308); GSK3β(S9); PTEN(S380); PDK1(S241); P70S6Kβ2(S371); mTOR(S2448); S6K(Ser235/236)/(Ser240/241)] conformation. The association of these variables with the Event Free Survival (EFS) was assessed using the χ2 test. A p value ≤0.05 was considered statistically significant. Furthermore, in order to measure the association level, the Relative Risk (RR) and the corresponding 95% Confidence Interval (95%CI) were calculated. Events were considered dead of complication (DOC), relapse and HSCT. Results. Seven out of the 23 patients presented an event: 5 relapses, 1 DOC and 1 HSCT. RT-PCR analysis of PTEN expression revealed that only one case did not show any product. Conversely, western blot analysis demonstrated that all patients showed total and phosphorylated PTEN proteins. Interestingly, we observed that total AKT protein was present in all the cases except one; the phosphorylated forms were detected as follows: AKT (T308) in 15 out of the 23 patients (65%), whereas none showed expression of AKT (S473). Surprisingly, we detected a statistically significant downregulation of total and phosphorylated mTOR and P70S6Kβ2 expression in eight, nine, ten and eleven out of 22 analyzed patients respectively. Downregulation or absent expression of both total and phosphorylated P70S6Kβ2 had a statistically significant impact on EFS showing a higher risk of events, when comparing those downregulated with those exhibiting phosphorylated (RR: 2,75; 95%CI: 1,25–6,01) and total protein (RR 3,33; 95%CI: 1,29–8,59) respectively. Moreover, downregulation of mTOR(S2448) confirmed the same pattern of higher risk of events (RR: 2,77; 95%CI: 1,08–7,07) comparing those downregulated with those exhibiting expression of phosphorylated protein. Conclusions. Our data for the first time have shown that the downregulation or absent expression of mTOR and P70S6Kβ2 is associated with a very poor outcome: 5 cases had very aggressive relapses (3 died for progressive disease); one child died during induction for complication related to aggressive disease (massive splenic hemorrhagic event) and one case underwent a matched-HLA familiar HSCT because of a high risk-MRD pattern. These preliminary findings need to be confirmed in a larger-population based study. Nevertheless our data identify new markers of aggressive and resistant disease, easily available at diagnosis, suggesting that mTOR and P70S6Kβ2, which play a crucial role as negative control in the PI3K/AKT cell signalling pathway, are needed to be evaluated in a future treatment plan design with specifically targeted drugs. Moreover, our data will be confirmed by the use of a reliable and robust method such as flow cytometry which will allow us to perform a sensitive and accurate measurement of single cell characteristics, emphasizing the intracellular signaling pathways of interest. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2154-2161 ◽  
Author(s):  
AG Turhan ◽  
FM Lemoine ◽  
C Debert ◽  
ML Bonnet ◽  
C Baillou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Promyelocytic Leukemia ◽  
Cell Sorting ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Pcr Analysis ◽  
Cell Populations ◽  
Rt Pcr ◽  
Hematopoietic Stem

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38-subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte- macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.

Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.)

Genome ◽  
1997 ◽  
Vol 40 (4) ◽  
pp. 570-581 ◽  
Author(s):  
Qing A. Yao ◽  
Ecaterina Simion ◽  
Manilal William ◽  
Joan Krochko ◽  
Ken J. Kasha
Keyword(s):  
Transgenic Plants ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Nuclear Dna ◽  
Active Agent ◽  
Biolistic Transformation ◽  
Pcr Analysis ◽  
Bar Gene ◽  
Isolated Microspores

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2–5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed β-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.Key words: barley, microspore, biolistic transformation, bialaphos, haploid, FISH.

scholarly journals Anti-Tumor Effects of BDH1 in Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Fei Han ◽  
Huanhuan Zhao ◽  
Jun Lu ◽  
Weina Yun ◽  
Lingling Yang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Poor Survival ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Worse Prognosis ◽  
Acute Myeloid

Dysregulation of ketone metabolism has been reported in various types of cancer. In order to find out its role in acute myeloid leukemia (AML) pathogenesis, we first analyzed the expression levels of 10 key genes involved in ketone metabolism in AML blasts and CD34+ hematopoietic stem cells (HSCs) from healthy donors. We found that the expression level of BDH1 was significantly lower in AML than in normal HSCs. The downregulation of BDH1 gene expression in AML cell lines as compared with normal HSCs was further confirmed with real-time RT-PCR. Analysis of TCGA and other database revealed that the downregulation of BDH1 was associated with worse prognosis in AML patients. In addition, we showed that overexpression of BDH1 inhibited the viability and proliferation of AML cells. In contrast, BDH1 knock-down promoted AML cell growth. Collectively, our results suggest the previously unappreciated anti-tumor role of BDH1 in AML, and low BDH1 expression predicts poor survival.

STAT5 Is a Negative Regulator of Megakaryopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1182-1182
Author(s):  
Lyndsay Drayer ◽  
Sandra Olthof ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Critical Role ◽  
Negative Regulator ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Erythroid Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Polyploid Cells

Abstract The transcription factor STAT5 plays a critical role in self-renewal and lineage commitment of hematopoietic stem cels (HSCs). We have recently shown in CB CD34+ cells that persistent activation of STAT5 results in enhanced self-renewal and induces erythroid differentiation, while myelopoiesis was severely impaired. In this study we analyzed the function of STAT5 during megakaryocyte (MK) differentiation. Using RNA interference we downregulated STAT5 expression in peripheral blood CD34+ cells. Cells were transduced with a lentiviral construct encoding eGFP and a short-hairpin RNA for STAT5 or with a control vector expressing YFP. Transduction efficiencies were determined by flow cytometric detection of eGFP/YFP and ranged from 40%–80%. Decreased STAT5 expression was verified by Western blot and quantitative RT-PCR (65% lower expression in STAT5 RNAi cells versus YFP+ control cells, p=&lt;0.01). To assess the effects of decreased STAT5 expression on the progenitor pool, the transduced populations were sorted and enumerated in colony assays. Downregulation of STAT5 significantly increased the number of MK progenitors (1.9-fold, p=0.02) and resulted in a decrease of erythroid progenitors (0.6-fold, p&lt;0.01), but did not affect the number of granulocyte/monocyte progenitors. Prospective isolation of immature and commited progenitors are currently being performed to distinguish if certain progenitor subsets are re-directed to the MK lineage by low STAT5 expression. Next, we analyzed the role of STAT5 during MK differentiation in unilineage suspension cultures. Transduced cells were cultured in serum-free medium containing TPO and SCF and scored for cell counts and expression of MK markers weekly. The percentage of transduced cells and their number did not differ when STAT5 RNAi cells were compared to controls, indicating that downregulation of STAT5 provides no proliferative advantage or disadvantage. However, the expression of glycoproteins IIb/IIIa (CD41) and Ib (CD42) was upregulated, and an increase in large, polyploid cells was observed in STAT5 RNAi cultures (15.2% polyploid cells versus 4.9% in control cells at day 7, p&lt;0.01). RT-PCR analysis of transcription factors predominantly expressed in MK and erythroid lineages revealed that Runx1 and Erg were increased in STAT5 RNAi cells, and the level of Scl was decreased compared to control cells. These observations correlate with the increased MK differentiation observed in STAT5 downregulated cells. Together, these data demonstrate that the expression level of STAT5 has important effects regarding the type of hematopoietic cell development, with high levels favouring erythroid development and low levels enhancing MK differentiation and maturation.

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