Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and highresolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.
IntroductionThe formation of new capillaries from pre-existing vessels (angiogenesis) appears to be essential for tumor growth and survival. [1][2][3][4][5][6] Whereas hypoxic conditions in expanding tumors often initiate a cascade of endothelial cell sprouting and formation of new vessels, tumor growth can be arrested by inadequate blood supply and a lack of metabolic exchange, often leading to tumor necrosis and regression. 7 Several tumor-derived, circulating angiogenesis inhibitors generated in vivo by proteolytic degradation have been recently identified (reviewed in Cao 8 and Marneros and Olsen 9 ). In particular, a 20-kDa C-terminal proteolytic fragment of collagen XVIII, termed endostatin, inhibits tumor growth in several animal models. [10][11][12][13][14][15] Although the mechanisms of action are incompletely understood, endostatin reportedly interacts with several endothelial cell-surface receptors that are critically involved in angiogenesis. Endostatin binds directly to the fibronectin receptor ␣ 5  1 16,17 and also interacts with ␣ v  3 and ␣ v  5 integrins. 17 In keeping with these interactions, endostatin inhibits endothelial cell binding to the ␣ v  3 -ligand gelatin 17 and also their binding to collagen type I in a dose-dependent manner. 18 Moreover, endostatin binds to heparan sulfate, [19][20][21][22] tropomyosin, 23 caveolin-1, 16 VEGFR-1 (vascular endothelial growth factor receptor-1; Flt-1), and VEGFR-2 (Flk-1) 24 and was recently shown to depend on E-selectin expression to be effective in vivo. 25 Interaction of endostatin with endothelial cells leads to a variety of downstream effects, [26][27][28] including inhibition of the Wnt/-catenin pathway, 29 and actin reorganization in endothelial cells. 16,[30][31][32][33] In fact, this wide variety of effects elicited by 1 endogenous inhibitor of angiogenesis was recently supported by 2 studies of global gene expression in endostatin-treated endothelial cells. 27,34 There is general agreement that the in vitro action of endostatin involves inhibition of endothelial cell migration, but less clear whether endostatin also affects endothelial cell apoptosis and proliferation. 6,12,26,[35][36][37][38][39] Furthermore, even less is known about how endostatin affects endothelial cell function in vivo, as readouts have been restricted to analysis of vascular density or blood flow in tumors, [12][13][14]35,40 or neovascularization of the chick allantoic membrane. 10,12,39 Moreover, studies that focused in more detail on endothelial cell behavior in vivo either failed to detect substantial effects of endostatin 41 or found subtle signs of impaired blood vessel maturation. 42 Finally, several studies failed to observe an antitumor effect of endostatin (reviewed in Marshall 43 ). Thus, there is an obvious need to more carefully analyze the effect of endostatin at the level of single endothelial cells during angiogenesis.Here, we describe the effect of endostatin in an in vivo model of human endothelial cell behavior in which the final stages of angio...
Development of resistance to cytarabine (AraC) is a major problem in the treatment of patients with acute myeloid leukemia (AML). Inactivation of deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We have identified inactive, alternatively spliced dCK forms in leukemic blasts from patients with resistant AML. Because these dCKspliced variants were only detectable in resistant AML, it was hypothesized that they might play a role in AraC resistance in vivo. In the current study, the biologic role of the alternatively spliced dCK forms in AraC resistance was further investi-
Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.
Resistance to cytarabine (AraC) is a major problem in treatment of patients with acute myeloid leukemia (AML). In contrast to in vitro AraC resistance, deoxycytidine kinase (dCK) mutations are rarely found in patients with refractory or relapsed AML. Previously we have demonstrated alternatively spliced dCK mRNA predominantly expressed in leukemic blasts from patients with resistant AML. In this study we investigated wild-type (wt) dCK expression and activity to elucidate the possible role of decreased dCK expression or activity in unresponsiveness to AraC in patients with AML. No alterations in dCK mRNA and protein expression or in dCK activity were detected between patients with clinically resistant vs. sensitive AML. In addition, wt dCK expression and activity were not reduced in leukemic blasts expressing alternatively spliced dCK forms as compared to blasts with only wt dCK. Also, no major differences in wt dCK expression and activity were observed between samples obtained from patients with AML and bone marrow or peripheral blood samples from healthy donors. These data implicate that in our patient group of refractory or relapsed AML cases, alterations in dCK expression and/or activity cannot explain unresponsiveness to chemotherapy including AraC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.