Abstract 2353
Hematopoiesis is maintained throughout life by the constant production of mature blood cells from hematopoietic stem cells (HSC). One mechanism by which the number of HSC is maintained ...is self-renewal, a cell division in which at least one of the daughter cells is a cell with the same functional potential as the mother cell. The mechanisms of this process are largely unknown. We have described cell lines that maintain self-renewal in culture. To study possible mechanisms and mediators involved in self-renewal, we performed co-cultures of HSC model cells: Lineage-negative Sca-1+ c-Kit+ (LSK) cells and HSC maintaining UG26–1B6 stromal cells. Microarray analyses were performed on cells prior to co-culture and cells sorted from the cultures. STEM clustering analysis of the data revealed that most changes in gene expression were due to early cell activation. Functional enrichment analysis revealed dynamic changes in focal adhesion and mTOR signaling, as well as changes in epigenetic regulators, such as HDAC in stromal cells. In LSK cells, genes whose products are involved in inflammation, Oxygen homeostasis and metabolism were differentially expressed after the co-culture. In addition, genes involved in the regulaton of H3K27 methylation were also affected. Interestingly, connective tissue growth factor (CTGF), which is involved in TGF-b, BMP and Wnt signaling, was upregulated in both stromal and LSK cells in the first day of co-culture. To study a possible extrinsic role of CTGF as a stromal mediator, we co-cultured siCTGF knockdown stromal cells with wild-type LSK cells. Since self-renewal requires cell division, we focused on cell cycle regulation of LSK cells. We found that knockdown of CTGF in stromal cells downregulates CTGF in LSK cells. In addition, knockdown of stromal CTGF downregulated Ccnd1, Cdk2, Cdkn1a (p21), Ep300 and Fos. On the other hand, decreased CTGF in stromal cells upregulates Cdkn1b (p27) and phosphorylation of Smad2/3. These results show that stromal CTGF regulates the cell cycle of LSK cells. On a functional level, we found that decreased stromal CTGF results in an increased production of MPP and myeloid colony-forming cells in 1-week co-cultures. We will present data showing whether and how a decrease in CTGF in stromal cells affects the maintenance of transplantable HSC. In summary, our current results indicate that reduced expression of CTGF in stromal cells regulates mediators of cell cycle and Smad2/3-mediated signaling in LSK cells, resulting in an increased production of myeloid progenitors.
No relevant conflicts of interest to declare.
Cancer pathogenesis involves tumor-intrinsic genomic aberrations and tumor-cell extrinsic mechanisms such as failure of immunosurveillance and structural and functional changes in the ...microenvironment. Using Myc as a model oncogene we established a conditional mouse bone marrow transduction/transplantation model where the conditional activation of the oncoprotein Myc expressed in the hematopoietic system could be assessed for influencing the host microenvironment. Constitutive ectopic expression of Myc resulted in rapid onset of a lethal myeloproliferative disorder with a median survival of 21 days. In contrast, brief 4-day Myc activation by means of the estrogen receptor (ER) agonist tamoxifen did not result in gross changes in the percentage/frequency of hematopoietic lineages or hematopoietic stem/ progenitor cell (HSPC) subsets, nor did Myc activation significantly change the composition of the non-hematopoietic microenvironment defined by phenotyping for CD31, ALCAM, and Sca-1 expression. Transcriptome analysis of endothelial CD45- Ter119- cells from tamoxifen-treated MycER bone marrow graft recipients revealed a gene expression signature characterized by specific changes in the Rho subfamily pathway members, in the transcription-translation-machinery and in angiogenesis. In conclusion, intra-hematopoietic Myc activation results in significant transcriptome alterations that can be attributed to oncogene-induced signals from hematopoietic cells towards the microenvironment, e. g. endothelial cells, supporting the idea that even pre-leukemic HSPC highjack components of the niche which then could protect and support the cancer-initiating population.
München, Technische Universität München, Diss., 2014
München, Technische Universität München, Diss., 2014
Munich, Technical University of Munich, Diss., 2014
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