[Show abstract][Hide abstract] ABSTRACT: The development of an independent blood supply by a tumor is essential for maintaining growth beyond a certain limited size and for providing a portal for metastatic dissemination. Host-derived endothelial cells (ECs) residing in and compromising the tumor vasculature originate via distinct processes known as sprouting angiogenesis and vasculogenesis. More recently ECs originating directly from the tumor cells themselves have been described although the basis for this phenomenon remains poorly understood. Here we describe in vitro conditions that allow lung and ovarian cancer cells to undergo a rapid and efficient transition into ECs that are indistinguishable from those obtained in vivo. A variety of methods were used to establish that the acquired phenotypes and behaviors of these tumor-derived ECs (TDECs) closely resemble those of authentic ECs. Xenografts arising from co-inoculated in vitro-derived TDECs and tumor cells were also more highly vascularized than control tumors; moreover, their blood vessels were on average larger and frequently contained admixtures of host-derived ECs and TDECs derived from the initial inoculum. These results demonstrate that cancer cells can be manipulated under well-defined in vitro conditions to initiate a tumor cell-to-EC transition that is largely cell-autonomous, highly efficient and closely mimics the in vivo process. These studies provide a suitable means by which to identify and perhaps modify the earliest steps in TDEC generation.
[Show abstract][Hide abstract] ABSTRACT: ATP half life. ATP levels were measured for the myc+/+ and myc−/−+wtMyc fibroblasts. The cells were incubated for the indicated times in the presence of 2-DG and oligomycin. A logarithmic curve was fit for each data set and the equation of the line was used to calculate the half life. Depicted is a representative experiment.
[Show abstract][Hide abstract] ABSTRACT: ECAR in rat fibroblasts. Extracellular acidification rates (ECARs) were calculated concurrently with OCR. ECAR is a surrogate measure of glycolysis and is expressed as a function of time. Inhibitors were injected at the times indicated by the arrows (injections: A-oligomycin, B-FCCP, C-2-DG, D-rotenone). A typical experiment, performed in triplicate wells is shown. The experiment was repeated on at least three occasions in replicates of four with similar results.
[Show abstract][Hide abstract] ABSTRACT: Western analysis for Myc expression. 5 µg of whole cell lysates from myc+/+, myc−/− and myc−/−wtMyc cells were used to perform Western analysis with the 9e10 anti-Myc antibody. β-actin is used as a loading control.
[Show abstract][Hide abstract] ABSTRACT: Although the c-Myc (Myc) oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS), the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc-/- fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC) are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell.