Lovastatin (LOV), the drug recently introduced to treat hypercholesteremia, inhibits the synthesis of mevalonic acid. The effects of LOV on the cell cycle progression of the human bladder carcinoma T24 cell line expressing activated p21ras were investigated. At a concentration of 2-10 microM, LOV arrested cells in G1 and also prolonged--or arrested a minor fraction of cells in--the G2 phase of the cell cycle; at a concentration of 50 microM, LOV was cytotoxic. The cytostatic effects were reversed by addition of exogenous mevalonate. Cells arrested in the cycle by LOV were viable for up to 72 hr and did not show any changes in RNA or protein content or chromatin condensation, which would be typical of either unbalanced growth or deep quiescence. The expression of the proliferation-associated nuclear proteins Ki-67 and p105 in these cells was reduced by up to 72% and 74%, respectively, compared with exponentially growing control cells. After removal of LOV, the cells resumed progression through the cycle; they entered S phase asynchronously after a lag of approximately 6 hr. Because mevalonate is essential for the posttranslational modification (isoprenylation) of p21ras, which in turn allows this protein to become attached to the cell membrane, the data suggest that the LOV-induced G1 arrest may be a consequence of the loss of the signal transduction capacity of p21ras. Indeed, while exposure of cells to LOV had no effect on the cellular content of p21ras (detected immunocytochemically), it altered the intracellular location of this protein, causing its dissociation from the cell membrane and translocation toward the cytoplasm and nucleus. However, it is also possible that inhibition of isoprenylation of proteins other than p21ras (e.g., nuclear lamins) by LOV may be responsible for the observed suppression of growth of T24 cells.

Key Points: Chloride channels are important for proper hydration of the airway surface. TMEM16A protein is an important component of calcium‐activated chloride channels. Interleukin‐4, a cytokine that induces mucous cell metaplasia, also upregulates calcium‐dependent chloride secretion in human bronchial epithelial cells. In bronchial epithelial cells treated with interleukin‐4, we found that TMEM16A protein becomes highly expressed in goblet but not in ciliated cells. Upregulation of TMEM16A by interleukin‐4 may be important for secretion and proper expansion of mucins. Abstract  The TMEM16A protein has a potential role as a Ca²⁺‐activated Cl⁻ channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o–). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short‐interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca²⁺‐dependent Cl⁻ transport. Following treatment for 24–72 h with interleukin‐4 (IL‐4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL‐4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A‐positive cells when epithelia were treated with IL‐4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL‐4, and that TMEM16A upregulation by IL‐4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3⁺ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4⁺ and CD8⁺ clones, and it down-regulates Ag recognition by CD8⁺ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca²⁺ mobilization.

Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with ¹⁸F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that ¹⁸F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy.