Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

Abstract

Evidence is presented that the marked stimulation of sugar It is important to note that this medium contains about 1.8 mM Ca2+, 0.8 mM Mg2+, and 0.9 mM Pi as normal medium components. Cells were grown in GM with 10% CS at 38°in 5% C02/95% air in 35-mm-diameter plates for experiments. Cultures were checked periodically for mycoplasma (11) and were uniformly negative. The Swiss 3T3 cells were used for most of these studies after it was noted that they showed larger responses to the various mitogens used.Uptake and Incorporation Assays. Procedures for assay of uptake of 2-deoxyglucose (dGlc) and 3-O-methylglucose (MeGlc) were essentially the same as previously described (12). Protein was determined by the method of Bradford (13). To determine the rate of incorporation of thymidine, [3H]thymidine was added directly to the medium (final concentration, 4 tCi/ml) 20 hr after addition of the mitogenic agent. Cells were incubated 4 hr with label, medium was aspirated, and the monolayers were washed three times with phosphate-buffered saline. The samples were precipitated with cold trichloroacetic acid and the radioactivity of the washed precipitate was measured in Aquasol. All data in which errors are given represent the mean of duplicate or triplicate samples ± average deviation from the mean. RESULTSEarly Experiments Linking Stimulation of dGlc Uptake with Precipitate Formation. In the course of experiments designed to test the effects of exogenous nucleotides on hormonal stimulation of sugar uptake in 3T3 cells, we observed a marked stimulation of dGlc uptake 3 or more hr after the addition of 0.5 mM UTP to cells in GM containing 10% CS. The stimulation was accompanied by appearance of a precipitate in the medium. This stimulation and precipitate formation were not seen in GM with 10% heat-inactivated CS or no CS and subsequently were found to be due to the presence of a nucleotide pyrophosphohydrolase activity in CS that generated PP1 from UTP in amounts large enough to form a calcium pyrophosphate precipitate. Stimulation of dGlc uptake by UTP could be mimicked by the addition of PPi alone (Table 1). However, this stimulation was seen only if the PP1 was allowed to form a precipitate in the medium; precipitation occurred only in media containing Ca2+. Addition of 1 mM EDTA to bind part of the Ca2+ retarded both the formation of the pre-

Increased Risk of Malignant Diseases After Surgery for Primary Hyperparathyroidism

“…It is possible that hyperparathyroidism may carry an increased risk of malignant disorders through its biochemical features. It is of interest in this context that both a high calcium level in vivo and in vitro and plasma from patients with hyperparathyroidism in vitro have been found to potentiate the mitogenic activity in bone marrow and lymphocyte mitogenesis (58)(59)(60)(61)(62). Another possible explanation is that hyperparathyroidism and the subsequent malignant disease have common etiologic factors, such as lack of calcium and/or vitamin D, as indicated above.…”

Mitogenic activity of hydroxyapatite: Requirement for somatomedin C

“…Only acid extracts of cell layer from cultures stimulated by HA crystals yield measurable amounts of Sm-C (2.6 ng/100 pg cell protein); therefore, the total measurahle amount of Sm-C in cultures stimulated by HA crystals was 9.7 ng/lOO pg cell protein. Barnes and Colowick (1977) described stimulation of 3H thymidine incorporation into mouse 3T3 cells in culture by precipitates of calcium phosphate. A similar growth stimulatory effect was observed with precipitates of calcium pyrophosphate (Rubin and Sanui, 1977).…”

Growth of human foreskin fibroblasts in a serum‐free, defined medium without platelet‐derived growth factor

“…Calcium stimulates DNA synthesis (in the absence of serum) in resting Balblc 3T3 cells (Dulbecco and Elkington, 1975), increases growth in serum-fed 3T3 cells (Dulbecco and Elkington, 1975;Rubin and Sanui, 1977), and stimulates the uptake of glucose (Barnes and Colowick, 1977) and uridine (Bowen-Pope et al, 1979) in Balblc 3T3 cells by influencing cellular magnesium homeostasis (Bowen-Pope et al, 1979). I t is possible that, in the absence of available calcium, PDGF manifests similar effects on M e homeostasis.…”

“…I t is possible that, in the absence of available calcium, PDGF manifests similar effects on M e homeostasis. Indeed, the time course and magnitude of sugar uptake by 3T3 cells is similar when stimulated by calcium, EGF, insulin or calf serum (Barnes and Colowick, 1977). Comparison of the effects of CaCI, to those of purified PDGF can now be performed under chemically defined conditions in the absence of serum.…”