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Abstract In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow and are in part regulated by the bone marrow microenvironment, called the stem cell niche. We have previously identified the bone marrow morphogen osteopontin (OPN), which is abundantly present in the bone marrow extracellular matrix, as a negative regulator of the size of the HSC pool under physiological conditions. Here, we study the impact of OPN on HSC function during aging using an OPN-knockout mouse model. We show that during aging OPN deficiency is associated with an increase in lymphocytes and a decline in erythrocytes in peripheral blood. In a bone marrow transplantation setting, aged OPN-deficient stem cells show reduced reconstitution ability likely due to insufficient differentiation of HSCs into more mature cells. In serial bone marrow transplantation, aged OPN−/− bone marrow cells fail to adequately reconstitute red blood cells and platelets, resulting in severe anemia and thrombocytopenia as well as premature deaths of recipient mice. Thus, OPN has different effects on HSCs in aged and young animals and is particularly important to maintain stem cell function in aging mice.

Cultivation of A549 non-small-cell lung carcinoma (NSCLC) cells in the presence of staurosporine (SSP) leads to a reduction or a lack of proliferation in a concentration-dependent manner. This inhibition of proliferation is accompanied by the generation of polyploid giant cancer cells (PGCCs) that are characterized by cell flattening, increased cell size, polyploidy, and polynucleation as determined by crystal violet staining, BrdU and DiI labelling, and flow cytometry as well as video time-lapse analysis. Continuous SSP treatment of A549 cells can preserve PGCCs for at least two months in a resting state. Upon removal of SSP, A549 PGCCs restart to divide and exhibit a proliferation pattern and cellular morphology indistinguishable from cells where PGCCs originally derived from. Thus, SSP-treated A549 cells represent a simple and reliable experimental model for the reversible generation of PGCCs and their subsequent experimental analysis.

Specialized blood cells are generated through the entire life of an organism by differentiation of a small number of hematopoietic stem cells (HSC). There are strictly regulated mechanisms assuring a constant and controlled production of mature blood cells. Although such mechanisms are not completely understood, some factors regulating cell cycle and differentiation have been identified. We have previously shown that Caspase-3 is an important regulator of HSC homeostasis and cytokine responsiveness. p21cip1/waf1 is a known cell cycle regulator, however its role in stem cell homeostasis seems to be limited. Several reports indicate interactions between p21cip1/waf1 and Caspase-3 in a cell type dependent manner. Here we studied the impact of simultaneous depletion of both factors on HSC homeostasis. Depletion of both Caspase-3 and p21cip1/waf1 resulted in an even more pronounced increase in the frequency of hematopoietic stem and progenitor cells. In addition, simultaneous deletion of both genes revealed a further increase of cell proliferation compared to single knock-outs and WT control mice, while apoptosis or self-renewal ability were not affected in any of the genotypes. Upon transplantation, p21cip1/waf1-/- bone marrow did not reveal significant alterations in engraftment of lethally irradiated mice, while Caspase-3 deficient HSPC displayed a significant reduction of blood cell production. However, when both p21cip1/waf1 and Caspase-3 were eliminated this differentiation defect caused by Caspase-3 deficiency was abrogated.

Small cell lung carcinomas (SCLCs) represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.