Expression of cyclin D1/2 in the lungs of strain A/J mice fed chemopreventive agents

Hanspeter Witschi 0 Imelda Espiritu 0 Marie Suffia 0 Kent E.Pinkerton 0 0 Center for Health and the Environment and Department of Molecular Biosciences, School of Veterinary Medicine, University of California , One Shields Avenue, Davis, CA 95616, USA 1To whom correspondence should be addressed Email: Male strain A mice were fed a diet containing chemopreventive agents. After 1 and 3 weeks on the diets, lung nuclear fractions were examined for expression of cyclin D1/2 with western blot analysis. In animals fed a diet containing a mixture of myoinositol and dexamethasone, a treatment found previously to be effective in preventing the development of tobacco smoke-induced lung tumors in A/J mice, cyclin D1/2 expression was reduced to 30-40% of control levels. A similar decrease in cyclin D1/2 expression was found when animals were fed either myoinositol or dexamethasone alone. Paradoxically, tobacco smoke by itself had a similar effect on cyclin D1/2 expression. On the other hand, several agents that had been previously found not to be effective against tobacco smoke carcinogenesis [phenethyl isothiocyanate, 1,4-phenylenebis(methylene)selenoisocyanate, N-acetylcysteine, acetylsalicylic acid, D-limonene and beta carotene] did not decrease cyclin D1/2 expression after 1 or 3 weeks of feeding. It was concluded that expression of cyclin D1/2 might be a potentially useful marker in the identification of chemopreventive agents for tobacco smoke and could be of some help in the evaluation of their effects. - Loss of cell cycle control is a key event in the progression from normal to cancerous tissue. Cyclins and the cyclindependent kinases (CDKs) are essential cell cycle regulators in normal cells. In cancer cells, their function is often overridden. Modulation of their activity might conceivably play an important role in cancer chemoprevention and therapy (1). Cyclin D1, a checkpoint control protein that acts at the midpoint of the transition from G1 to S, has received particular attention. In many human tumors the protein is over-expressed. It has been postulated that drugs capable of inhibiting cyclin D1 expression could be useful in cancer chemoprevention (2,3). It has recently been shown in a rat mammary tumor model that tamoxifen, a proven chemopreventive agent, decreases expression of cyclins D1 and E (4). Another putative chemopreventive agent, epigallocatechin-3-gallate, decreases cyclin D1 expression in human breast and epidermoid carcinoma cells (5,6). Studies with transformed human bronchial epithelial cells have shown that a third group of chemopreventive agents, Abbreviations: ASA, acetylsalicylic acid (aspirin); CDK, cyclin-dependent kinases; NAC, N-acetylcysteine; PEITC, phenethyl isothiocyanate; pXSC, 1,4phenylenebis(methylene)selenoisocyanate; TSP, total suspended particulates. retinoids, markedly reduce expression of cyclins D and E and of cdk2 and of cdk4, presumably due to increased proteolysis at the post-translational level. On the other hand, expression of p27 was eventually increased, whereas p21 was somewhat decreased and p16 did not change (79). During the last few years, we have conducted a series of studies in which we examined the effects of chemopreventive agents on the development of tobacco smoke-induced lung tumors in strain A/J mice (1013). In these studies we found that a combination of myoinositol and dexamethasone, fed in the diet, proved to be an effective chemopreventive regimen. On the other hand, several other agents such as phenethyl isothiocyanate (PEITC), 1,4-phenylenebis (methylene) selenoisocyanate (pXSC), green tea, aspirin, N-acetylcysteine (NAC) or D-limonene, although previously having been shown to be effective against selected constituents of tobacco smoke, were largely ineffective when tested against the full complex mixture of cigarette smoke. It was therefore of interest to examine cyclin D1 expression in the lungs of animals fed successful or ineffectual chemopreventive diets and to see whether cyclin D expression could serve as a biomarker of effect. Materials and methods Animals Male strain A/J mice, 68 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME). Randomly chosen animals were sent to the Comparative Pathology Laboratory, UC Davis, for a standard rodent health surveillance screen. No evidence for infectious disease (pathogenic agents) or presence of parasites or ova in pelage and cecum were reported. Histopathology was not processed as no significant lesions were noted. Serology was negative for mouse hepatitis virus, Sendai virus, Reovirus type 3, pneumonia virus, parvo, ectromelia and mycoplasma pulmonis. The animals were housed, four to a cage, on Teklad bedding in polypropylene cages with tightly fitting wire screen lids. At all times during the experiment, including during smoke exposure, water and the test diets were provided ad libitum. Materials Anti-cyclin D1/2, clone 5D4 was purchased from Upstate Biotechnology (Lake Placid, NY) and anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA). PEITC, NAC, acetylsalicylic acid (ASA), myoinositol, dexamethasone, D-limonene and corn oil were obtained from Sigma Chemical Co. (St Louis, MO). The AIN-76A test diet was purchased in powdered form from Dyets (Bethlehem, PA) and consisted of 20% casein, 0.3% DL-methionine, 15% corn starch, 55% sucrose, 5% cellulose, 3.5% mineral mix, 1% vitamin mix and 0.2% choline bitartrate. The organoselenium compound pXSC was synthesized according to the method originally described by El-Bayoumy et al. (14). Purity and spectral characteristics were the same as described before (12). Test diets containing the chemopreventive agents were prepared fresh every other week by adding the appropriate amounts of the ingredients plus 50 ml of corn oil/kg of diet. The diets were mixed thoroughly in a Hobart blender and stored at 4C until used. All other materials were of the highest obtainable grade. Tobacco smoke exposure Mice were exposed to a mixture of 89% cigarette sidestream and 11% mainstream smoke generated from burning Kentucky 1R4F reference cigarettes in the exposure system as described before (15,16). Exposure was for 6 h a day, 5 days a week to an average concentration of 130 mg total suspended particulate matter (TSP) per cubic meter air. Western blot analysis All animals were killed by pentobarbital overdose. Protein from lungs was prepared as described by Sabourin et al. (17). Lungs were homogenized gently with a teflon-glass homogenizer in buffer consisting of 50 mM TrisHCl pH 7.5, 150 mM NaCl, 50 mM NaF, 1.0 M EDTA, 1.0 M Na3VO4,, 1.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonide fluoride, 25 g/ml aprotinin, 25 g/ml leupeptin and 7 g/ml pepstatin. The homogenate was centrifuged at 14 000 g for 15 min and protein concentration measured by using the BioRad assay (Bio-Rad, Richmond, CA). Fifty to 100 g of protein were mixed with loading buffer (187.5 mM Tris buffer, pH 6.8, 6% SDS, 30% (...truncated)