Age-specific biological and molecular profiling distinguishes paediatric from adult acute myeloid leukaemias

Acute myeloid leukaemia (AML) affects children and adults of all ages. AML remains one of the major causes of death in children with cancer and for children with AML relapse is the most common cause of death. Here, by modelling AML in vivo we demonstrate that AML is discriminated by the age of the cell of origin. Young cells give rise to myeloid, lymphoid or mixed phenotype acute leukaemia, whereas adult cells give rise exclusively to AML, with a shorter latency. Unlike adult, young AML cells do not remodel the bone marrow stroma. Transcriptional analysis distinguishes young AML by the upregulation of immune pathways. Analysis of human paediatric AML samples recapitulates a paediatric immune cell interaction gene signature, highlighting two genes, RGS10 and FAM26F as prognostically significant. This work advances our understanding of paediatric AML biology, and provides murine models that offer the potential for developing paediatric specific therapeutic strategies

Doxorubicin induces the DNA damage response in cultured human mesenchymal stem cells

Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562. Phosphorylation of H2AX, Chk1, and RPA2 was more strongly activated in K562 cells than in hMSCs, at equivalent doses of doxorubicin. hMSCs were relatively resistant to doxorubicin such that, following exposure to 15 μM doxorubicin, the level of cleaved caspase-3 detected by western blotting was lower in hMSCs compared to K562 cells. Flow cytometric analysis of cell cycle progression demonstrated that exposure to doxorubicin induced G2/M phase arrest in hMSCs, while 48 h after exposure, 15.6 % of cells were apoptotic, as determined from the percentage of cells having sub-G1 DNA content. We also show that the doxorubicin sensitivity of hMSCs isolated from a healthy donor was comparable to that of hMSCs isolated from a chronic lymphocytic leukemia patient. Overall, our results demonstrate that high doses of doxorubicin induce the DNA damage response in hMSCs, and that cultured hMSCs are relatively resistant to doxorubicin.Deposited by bulk impor

Activation of dna damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to gamma-irradiation and cisplatin, key DNA damage responses have been characterized in hMSCs from normal adult donors. Cisplatin and gamma-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, gamma H2AX staining decreased by 24 h following gamma-irradiation. gamma-irradiation arrested hMSCs in the G 1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and gamma-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared with peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and gamma-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment

Doxorubicin induces the dna damage response in cultured human mesenchymal stem cells

Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562. Phosphorylation of H2AX, Chk1, and RPA2 was more strongly activated in K562 cells than in hMSCs, at equivalent doses of doxorubicin. hMSCs were relatively resistant to doxorubicin such that, following exposure to 15 mu M doxorubicin, the level of cleaved caspase-3 detected by western blotting was lower in hMSCs compared to K562 cells. Flow cytometric analysis of cell cycle progression demonstrated that exposure to doxorubicin induced G2/M phase arrest in hMSCs, while 48 h after exposure, 15.6 % of cells were apoptotic, as determined from the percentage of cells having sub-G1 DNA content. We also show that the doxorubicin sensitivity of hMSCs isolated from a healthy donor was comparable to that of hMSCs isolated from a chronic lymphocytic leukemia patient. Overall, our results demonstrate that high doses of doxorubicin induce the DNA damage response in hMSCs, and that cultured hMSCs are relatively resistant to doxorubicin

Activation of dna damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to gamma-irradiation and cisplatin, key DNA damage responses have been characterized in hMSCs from normal adult donors. Cisplatin and gamma-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, gamma H2AX staining decreased by 24 h following gamma-irradiation. gamma-irradiation arrested hMSCs in the G 1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and gamma-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared with peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and gamma-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment

ROCK activity and the Gβγ complex mediate chemotactic migration of mouse bone marrow-derived stromal cells

Bone marrow-derived stromal cells (BMSCs), also known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. Despite the importance of BMSC migration for their potential therapeutic uses, the mechanisms and signalling governing stem cell migration are still not fully elucidated. Methods: We investigated and detailed the effects of MCP-1 activation on BMSCs by using inhibitors of G protein-coupled receptor alpha beta (GPCR αβ), ROCK (Rho-associated, coiled-coil containing protein kinase), and PI3 kinase (PI3K). The effects of MCP-1 stimulation on intracellular signalling cascades were characterised by using immunoblotting and immunofluorescence. The effectors of MCP-1-mediated migration were investigated by using migration assays (both two-dimensional and three-dimensional) in combination with inhibitors. Results: We established the kinetics of the MCP-1-activated signalling cascade and show that this cascade correlates with cell surface re-localisation of chemokine (C motif) receptor 2 (CCR2) (the MCP-1 receptor) to the cell periphery following MCP-1 stimulation. We show that MCP-1-initiated signalling is dependent on the activation of βγ subunits from the GPCR αβγ complex. In addition, we characterise a novel role for PI3Kγ signalling for the activation of both PAK and ERK following MCP-1 stimulation. We present evidence that the Gβγ complex is responsible for PI3K/Akt, PAK, and ERK signalling induced by MCP-1 in BMSCs. Importantly, we found that, in BMSCs, inhibition of ROCK significantly inhibits MCP-1-induced chemotactic migration, in contrast to previous reports in other systems.Conclusions: Our results indicate differential chemotactic signalling in mouse BMSCs, which has important implications for the translation of in vivo mouse model findings into human trials. We identified novel components and interactions activated by MCP-1-mediated signalling, which are important for stem cell migration. This work has identified additional potential therapeutic targets that could be manipulated to improve BMSC delivery and homing.Science Foundation IrelandNational Breast Cancer Research Institut

Critically important antimicrobial resistant Enterobacteriaceae in Irish farm effluent and their removal in integrated constructed wetlands

peer-reviewedThis study investigated the ability of Integrated Constructed Wetlands (ICWs) to remove critically important antimicrobial resistant organisms (AROs) from farm wastewater. Influent samples from the untreated farm waste and effluent samples taken at the end of the ICW system were collected monthly from four ICWs, serving four different farm types (suckler, dairy, dairy & poultry and pig). Using selective media to screen for the presence of carbapenemase resistant organisms, plasmid mediated and AmpC β-Lactamase producing organisms (ESBL/pAmpC) and fluoroquinolone resistant organisms, a total of 82 AROs were obtained with the majority being E. coli (n = 79). Statistically significant were the differences on the number of AROs isolated from influent (higher) compared to effluent, as well as a seasonal effect, with less AROs recovered during winter in comparison to other seasons (P < 0.05). On the other hand, there was no significant differences in the recovery of AROs on different farms. The majority of isolates from each of the farms (99%) were multi drug resistant, with 65% resistant to seven or more antimicrobials. A high incidence of tetracycline, trimethoprim/sulfamethoxazole, and ampicillin resistance was common to the isolates from all four farms but there were differences in ESBL levels with 63% of the isolates recovered from Farm 4 (piggery) being ESBLs compared to 18%, 36% and 4.5% recovered from Farms 1 (suckler), 2 (dairy) and 3 (dairy & poultry), respectively. No carbapenemase producing organisms were isolated. Our results showed that ICWs are effective in removing critically important AROs from farm wastewater on all four farm types