CD133 Is a Marker of Bioenergetic Stress in Human Glioma

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho0 or ρ0, are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these ρ0 cells display the ability to form “tumor spheroids” in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, ρ0 cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to ρ0 cells resulting in stable trans-mitochondrial “cybrid” clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised

Expression of glucagon sensitivity by transformed MDCK cells normally unresponsive to glucagon: Early commitment to differentiation

A cloned line of canine kidney cells (MDCK) transformed with Harvey murine sarcoma virus, in contrast to the parental, untransformed line, expressed glucagon sensitivity only under controlled culture conditions. The glucagon sensitivity of transformed MDCK cells appeared after 10 days of culture if plated at less than 100,000 cells/dish or after 3 days if cells were plated at greater than 300,000 cells/dish. As there was no effect of conditioned medium from glucagon-sensitive cells on insensitive cells, media components seemed not be involved in this phenomenon. Glucagon sensitivity appeared more readily in defined as opposed to serum-containing medium. In fact, as little as 2% fetal bovine serum inhibited the expression of glucagon sensitivity when included in defined medium over the course of the experiment. Furthermore, when transformed MDCK cells were exposed to serum for only the first 24 hr of culture, glucagon sensitivity on day 11 was identical to that of cells exposed to serum throughout the entire experiment. In contrast, exposure to serum later in culture (days 4-8) had no inhibitory effect on the expression of glucagon sensitivity on day 11. The data suggest that differentiation, or glucagon sensitivity, occurs when transformed, glucagon-insensitive cells achieve a critical high density and that differentiation is sensitive to inhibition by serum only during the first 24 hr of culture.link_to_subscribed_fulltex

Glucagon sensitivity of transformed MDCK cells; early commitment to differentiate

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Characterization of hormone-sensitive Madin-Darby canine kidney cells

Cultured cells, especially the established lines, provide a continuous and homogeneous system for studying hormone action in intact cells. The cell line known as Madin–Darby canine kidney (MDCK) cells, derived from normal dog kidney more than 20 years ago, is ideal for this type of research, because it retains differentiated functions in culture and, in addition, responds to several hormones, including glucagon, vasopressin, β-adrenergic agonists, and prostaglandins. This chapter deals mainly with the optimal culture conditions for maintaining hormone responsiveness, the measurement of intracellular cyclic adenosine monophosphate (AMP). The chapter also discusses the characteristics of several types of hormone sensitivity in MDCK cells and concludes that MDCK cells have been used as a model system for studying hormone regulation of kidney functions. The availability of a differentiated, hormone-responsive cell line facilitates the studies of the cascade of biochemical events subsequent to cyclic AMP elevation and its correlation with specific kidney functions.link_to_subscribed_fulltex

Decreased potency of glucoagon on transformed-induced MDCK cells does not reflect an alteration of adenylate cyclase components

The selective loss of glucagon sensitivity of transformed MDCK cells can be restored by differentiation inducers, a process which requires RNA and protein synthesis and glycosylation. Although the glucagon dose-response curve of normal MDCK cells resembled that of liver and kidney (K(act) = 10 nM), the transformed-induced cells were 10-fold less sensitive to the hormone [activation constant (K(act)) = 100 nM]. Additionally, the stimulation of cAMP synthesis by a glucagon fragment (glucagon 1-27) in transformed-induced cells was greatly reduced compared to normal cells. The adenylate cyclase regulatory components of transformed-induced MDCK cell membranes seemed unaltered compared to the parental line. both contained equivalent amounts of cholera and pertussis toxin substrates, and soluble extracts were equally capable of reconstituting isoproterenol responsiveness of S49 cyc - membranes. However, membrane fusion studies demonstrated that the glucagon sensitivity of transformed-induced membranes could not be reconstituted with heterologous membranes. When donor transformed-induced membranes (with inactivated adenylate cyclase) were fused with acceptor HeLa membranes (normally unresponsive to glucoagon and prostaglandin E), such hybrids were unresponsive to glucagon, although responsiveness to prostaglandin E was evident. Parallel hybrids with normal MDCK membranes were responsive to both glucagon and prostaglandin E. This difference could not be explained by an inhibitory effect of transformed-induced membranes on receptor-adenylate cyclase coupling under the fusion conditions: the ability of these membranes to serve as an acceptor for the reconstitution of vasoactive intestinal peptide responsiveness was identical to that of normal MDCK cells. The data suggest that the glucagon sensitivity induced in transformed MDCK cells differs significantly from that of the parental line. However, these differences cannot be explained by alterations of transformed-induced membrane components relevant to the coupling of hormone receptors to adenylate cyclase.link_to_subscribed_fulltex

[30] Induction of glucagon responsiveness in transformed MDCK cells unresponsive to glucagon

This chapter discusses a cloned line of Madin–Darby canine kidney (MDCK) cells, which were transformed with Harvey murine sarcoma virus. This line is maintained in continuous culture under the same conditions as the parental MDCK cells in Dulbecco's MEM, 5% fetal bovine serum, 5% CO2/95% air, with 80% humidity. The morphology of this transformed line is more fibroblastic than that of normal cells; in addition, a 21,000 Da protein (p21) coded by the virus has been identified on the inner surface of the plasma membrane of this transformed line. The growth characteristics of transformed MDCK cells are similar to normal cells; both grow equally well in serum free media. Like normal MDCK cells, the adenylate cyclase of the transformed line responds to a variety of hormones. However, transformation results in a selective loss of glucagon responsiveness because of the absence of glucagon binding sites at the cell surface. Thus, this cell line represents a good model system to examine factors that regulate the expression of differentiated functions.link_to_subscribed_fulltex

The effect of viral transformation on prostaglandin production depends on cell type

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