Growth- and Density-Dependent Inhibition of Deoxyglucose Transport in Balb 3T3 Cells and Its Absence in Cells Transformed by Murine Sarcoma Virus

Abstract

The earliest measurable parameter that shows density-dependent inhibition is the uptake of [IHfdeoxyglucose by Balb 3T3 cells. The rate decreases even during the exponential phase of growth and reaches a minimum, about 8-to 10-times lower than maximum, as the culture approaches the saturation density. Cells transformed by murine sarcoma virus fail to show either growth-dependent or density-dependent inhibition of deoxyglucose uptake. Treatment with dibutyryl cyclic AMP in the presence of theophylline results in premature cessation of growth and in an arrest in the decline of deoxyglucose transport. Culture in serum-deficient medium also produces rapid inhibition of growth at low cell density, but these cultures exhibit a markedly decreased rate of deoxyglucose uptake.Growth in vitro of "normal" cells is usually well controlled in that its extent, expressed as saturation density, is limited. Several factors are thought to be involved in determining the saturation density; among them are (i) topoinhibition brought about by cell-to-cell proximity and (ii) availability of smallmolecular-weight nutrients from the medium, including several factors present in the serum normally incorporated in the medium. Cells transformed by oncogenic viruses are not subject to the growth restriction brought about by crowding or lack of serum. Stoker has described the transformed malignant cells as "asocial" cells (1). Tomkins and his coworkers have considered the behavior of virus-transformed cells as analogous to the "relaxed control" of RNA synthesis in bacteria and have proposed that a "pleiotropic control mechanism," regulating several metabolic activities in eukaryotic cells, is "relaxed" in tranformed cells (2). Holley has proposed that growth of cells is controlled by modification of transport sites that regulate the availability of critical nutrients (3). The putative modifiers of the transport sites are hormones such as insulin (4), or growth factors such as the ones found in serum (5-7).The cell-surface membrane has been implicated in the altered phenotype exhibited in growth, morphology, high lectin agglutinability, modification of glycopeptides, appearance of new antigens on the cell surface, and high hexose transport, which are considered to be characteristics of malignant transformed cells. During an investigation of increased uptake of 2-deoxy-D-glucose demonstrated by cells transformed by the Harvey strain of murine sarcoma virus (H-MSV) (8), which led to the development of a biochemical assay of the transforming activity of murine sarcoma viruses (9), we observed marked changes in the ability of normal cells to transport deoxyglucose. Cells transformed by H-MSV, on the other hand, fail to exhibit these changes. The data presented here indicate that the ability of cells to transport glucose is related to the extent of growth (density) of the culture and suggest to us the possibility that this relation may have growth regulatory features. MATERIALS AND METHODSCells and Their Culture. Balb 3T3 cells were obtaine...

Cyclic AMP, a nonessential regulator of the cell cycle.

“…Transformed cells have a lower cAMP content than untransformed cells (1,5). Exogenous cAMP analogs or the induction of endogenous cAMP slows or stops growth of some cells (6)(7)(8)(9)(10). Proliferation of contact-inhibited cells, induced by refeeding or proteolytic treatment, is prevented by cAMP analogs (7,11).…”

Inhibition by Ethidium Bromide of the Establishment of Infection by Murine Sarcoma Virus

“…The conditions of growth of Harvey MSV (containing MLV) have been described (Bose & Zlotnick, 1973).…”

“…The mouse cell line Balb 3T3 was cultured as described previously (Bose & Zlotnick, 1973). The population doubling time (20 to 22 h) and saturation density (< 5 × Io4/cm2) was monitored continuously and at least once in 2 months new cultures were started from stocks stored in liquid N2.…”

Changes in microsomal Na+,K+-,Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells.

“…Transport activities for deoxyglucose (1,5) and non-metabolizable amino acids (5,12) in contact-inhibited cells are reduced with increases in cell density, but no changes occur in transformed cells that do not show contact inhibition. Ouabain-sensitive uptake of 86Rb+, as a K+ analogue, also decreases with increasing density of untransformed cells (10).…”

“…Contact inhibition of cell growth has been reported to be associated with changes in plasma membrane functions (1,5,12,15). Transport activities for deoxyglucose (1,5) and non-metabolizable amino acids (5,12) in contact-inhibited cells are reduced with increases in cell density, but no changes occur in transformed cells that do not show contact inhibition.…”