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Human intervention trials have suggested that supplemental β-carotene resulted in more cancer in smokers, whereas it was protective in non-smokers. However, the mechanisms underlying these effects are still unknown. The aim of this study was to evaluate the effects of an association of cigarette smoke condensate (tar) and β-carotene on DNA oxidative damage and molecular pathways involved in cell cycle progression and apoptosis in cultured cells. In RAT-1 fibroblasts, tar caused increased levels of 8-hydroxyl-2′-deoxyguanosine (8-OHdG) and this effect was enhanced by the concomitant presence of β-carotene (0.5–4.0 µM) in a dose- and time-dependent manner. In contrast, β-carotene alone did not significantly modify it. Fibroblasts treated with tar alone decreased their cell growth with respect to control cells through an arrest of cell cycle progression in the G0/G1 phase and an induction of apoptosis. These effects were accompanied by an increased expression of p53, p21 and Bax and by a decreased expression of cyclin D1. In contrast, fibroblasts treated with tar and β-carotene, after an initial arrest of cell growth at 12 h, re-entered in cell cycle and were unable to undergo apoptosis at 36 h. Concomitantly, their p53 expression, after an increase at 12 h, progressively returned at basal levels at 36 h by a mechanism independent of Mdm2. Such a decrease was followed by a decrease in p21 and Bax expression and by an increase in cyclin D1 expression. Moreover, the presence of the carotenoid remarkably enhanced cyclooxygenase-2 expression induced by tar. During tar treatment, a depletion of β-carotene was observed in fibroblasts. The effects of tar and β-carotene on 8-OHdG levels, cell growth and apoptosis were also observed in Mv1Lu lung, MCF-7 mammary, Hep-2 larynx and LS-174 colon cancer cells. This study supports the evidence for potential detrimental effects of an association between β-carotene and cigarette smoke condensate.

Although epidemiologic studies have demonstrated that a high intake of vegetables containing β-carotene lowers the risk of cancer, recent intervention studies have revealed that β-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that β-carotene may act as a pro- or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to β-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, β-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by β-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, α-tocopherol minimized the effects of β-carotene on cell growth. These data provide evidence that β-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells. © 2001 Wiley-Liss, Inc.

Extracellular magnesium (Mg) depletion inhibits the growth of the HC11 normal mammary epithelial cells. In this study we found that an acute increase in extracellular Mg generally exerts a positive effect on the growth of these cells. We also isolated and characterized two derivatives adapted to grow and proliferate at nonphysiological concentration of Mg. The growth properties of the HC-LMg cells at 25 μM Mg were comparable to those of the parental HC11 cells in the regular medium (0.5 mM Mg) despite an increased expression of the CDK inhibitor p27Kip1. They also showed a reduced dependence from serum to grow. The HC-HMg cells have been adapted to grow and proliferate at an increased (45 mM) Mg concentration. Cell total Mg content was 19.6, 9.7, and 20.1 nmol/mg protein in the HC11, HC-LMg, and HC-HMg cells, respectively. Thus, we have isolated derivatives of normal epithelial cells which are able to maintain Mg content in a physiological range in the face of different extracellular concentration gradients and will be a valuable tool for further studies on the regulation of Mg homeostasis in eukaryotic cells.

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF-7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg²⁺ (from 0.5 to 0 mM) and the growth rates were determined by [³H]-thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg²⁺ depletion whereas the MCF-7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg²⁺ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF-7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin-dependent kinase inhibitor p27^Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF-7 cells. We also demonstrated that Mg²⁺ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg²⁺-restricted HC11 cells are still responsive to the epidermal growth factor (EGF)- and insulin-mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg²⁺ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27^Kip1. J. Cell. Physiol. 180:245–254, 1999. © 1999 Wiley-Liss, Inc.

Rat spleen lymphocytes (RSL) incubated at 37°C in Mg-free medium (0-transconditions) exibited Mg2+efflux with apparent velocity of 0.2 nmol/mg protein/min. After 30 min, this process accounted for the mobilization of about 15% of cell total Mg2+. Half of the Mg2+efflux depended on extracellular Na+and was stimulated by cAMP. IFN-α significantly enhanced Mg2+efflux under0-transconditions as well as in the presence of physiological extracellular Mg2+. Pretreatment of RSL with indomethacin completely abolished IFN-α-induced Mg2+efflux, suggesting a crucial role for cyclo-oxygenase-dependent arachidonate metabolism. On the other hand, pretreatment of RSL with the PKA inhibitor (Rp)8-Br-cAMPS prevented IFN-α stimulation of Mg2+efflux, indicating the involvement of cAMP. Consistently, both IFN-α and exogenous PGE1increased cAMP from 50 to 125 pmol/mg protein. Altogether these results show that IFN-α stimulates Mg2+efflux by activating arachidonate metabolism and synthesis of prostaglandins. By influencing adenylcyclase activity, PGEs can eventually promote cAMP-dependent Mg2+efflux, possibly through the activity of a Na–Mg antiport. In RSL, therefore, magnesium movements can be under the control of IFN-α and, perhaps, of other cytokines, suggesting the involvement of Mg2+in cell response to receptor-mediated stimuli.

Extracellular ATP causes 40% stimulation of Mg2+efflux from Ehrlich ascites tumor cells (EATC) incubated under0-transconditions. ATP also causes a threefold increase of arachidonic acid (AA) metabolite release from [3H]AA-preloaded EATC, indicating that, under these experimental conditions, it induces phospholipase A2(PLA2) activation. ATP-induced Mg2+efflux can be prevented by cyclooxygenase inhibition with indomethacin or lysine acetylsalicylate, but not by 5-lipooxygenase inhibition with BWA4C. Mg2+efflux is also directly stimulated by exogenous AA in a concentration-dependent manner. This phenomenon involves PKA as it is virtually abolished by the specific inhibitor 8-bromoadenosine-3′,5′-cyclic monophosphothioate. While stimulating Mg2+efflux, exogenous AA also increases cAMP content of EATC and this effect can be prevented by cyclooxygenase inhibition. Measurements in mag-fura-2-loaded EATC reveal that stimulation of Mg2+efflux does not correlate with significant fluctuations of [Mg2+]i. This suggests that Mg2+efflux is compensated for by mobilization of bound Mg2+, leaving [Mg2+]iunaltered. Altogether these data indicate that Mg2+efflux can be modulated by extracellular stimuli capable of activating PLA2. This modulation is triggered by cyclooxygenase products which, by activating adenylcyclase, determine an elevation of cAMP. This intracellular messenger upregulates Na-dependent Mg2+efflux.

Commentary to:Human mitochondrial Mrs2 protein promotes multidrug resistance in gastric cancer cells by regulating p27, cyclin D1 expression and cytochrome C releaseYu Chen, Xufeng Wei, Pengfei Yan, Ying Han , Kai-Chun Wu, Shiren Sun, Daiming Fan

Comment on: Sahni J, et al. Cell Cycle 2010; 9:3565-74.