Allium chemistry: synthesis, natural occurrence, biological activity, and chemistry of Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products.

Syntheses are reported for gamma-glutamyl Se-methylselenocysteine (Sa), selenolanthionine (16), Se-1-propenylselenocysteine (Gd), Se-2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-propynyl-L-selenocysteine (6f). Oxidation of 8a and Se-methylselenocysteine (Ga) gives methaneseleninic acid (24), characterized by X-ray crystallography, and dimethyl diselenide (25). Oxidation of Se-2-propenyl-L-selenocysteine (6c) gives allyl alcohol and 3-seleninoalanine (22). Compound 22 is also formed on oxidation of 16 and selenocystine (4). Oxidation of 6d gives 2-[(E,Z)-1-propenylseleno]propanal (36). These oxidations occur by way of selenoxides, detected by chromatographic and spectroscopic methods. The natural occurrence of many of the Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products is discussed. Three homologues of the potent cancer chemoprevention agents 6a and 6c, namely 6d-f, were evaluated for effects on cell growth, induction of apoptosis, and DNA-damaging activity using two murine mammary epithelial cell lines. Although each compound displays a unique profile of activity, none of these compounds (Gd-f) is likely to exceed the chemopreventive efficacy of selenocysteine Se-conjugates Ga and 6c.

Molecular mechanisms associated with Se-allylselenocysteine regulation of cell proliferation and apoptosis.

Se-allylselenocysteine (ASC) has been shown to inhibit mammary carcinogenesis in vivo and cell growth in vitro. However, little is known about the molecular events that account for these effects. The goal of the present study was to use a mouse hyperplastic mammary epithelial cell line, TM12, to investigate the underlying mechanism(s) associated with ASC regulation of cell proliferation and apoptosis. Cells were treated with 50 microM ASC and assessed after 3, 6 and 12 h of exposure. A significant inhibition of cell proliferation, as measured by BrdU incorporation into DNA, was observed within 3 h of ASC treatment. This inhibitory effect was slightly magnified at the later time points. The induction of apoptosis was also rapid, and progressed from a 1.3-fold increase at 3 h to a 4.4-fold increase at 12 h. Consistent with these cellular events, the levels of phosphorylated Rb protein were greatly reduced at all times points. The other accompanying changes included increases in P53, P21 and P27. Collectively, the results demonstrate for the first time that ASC is able to cause an immediate response in the expression of cell cycle regulatory proteins that favor an arrest in proliferation and an augmentation in apoptosis.

Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea.

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague-Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3 mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r = 0.05, p > 0.80), whereas there was a significant correlation in ductal epithelium (r = 0.86, p = 0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p < 0.03). All carcinomas from vorozole-treated rats expressed PR (2.5-60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r = 0.42, p = 0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.

Control of rat mammary epithelium proliferation by conjugated linoleic acid.

Past research showed that mammary gland morphogenesis in the pubescent rat was retarded by the feeding of conjugated linoleic acid (CLA). A major objective of the present study was to examine the proliferative activity and the expression of cell cycle regulatory proteins in the developing mammary epithelium of rats fed a mixture of CLA isomers (primarily as free fatty acid c9, t11-CLA and t10,c12-CLA) or a highly enriched natural source of c9,t11-CLA (as triacylglycerol in butterfat). In both experiments, the diets, with or without CLA, were started at weaning and continued for four weeks. The two CLA preparations were equally effective in suppressing bromodeoxyuridine labeling and the expression of cyclin D1 and cyclin A (determined by immunohistochemistry) in the terminal end buds and alveolar clusters of the mammary epithelium while it undergoes extensive ductal branching during pubescence. There was a trend of an increase, although not statistically significant, in the proportion of cells expressing the p16 and p27 cdk inhibitors. A separate experiment was designed to evaluate the effect of c9,t11-CLA (as a free fatty acid of > 90% purity) treatment on the rate of proliferation of the mammary epithelium as the animal matured from weanling to adult. The bromodeoxyuridine labeling data indicated that the mammary epithelium appeared to lose its sensitivity to CLA control of proliferation as it completely filled the fat pad and became quiescent. These observations suggest that the responsiveness of mammary epithelial cells to CLA intervention may be dependent on their proliferative status.

In vitro effects of Se-allylselenocysteine and Se-propylselenocysteine on cell growth, DNA integrity, and apoptosis.

Two previously unevaluated selenium compounds, Se-allylselenocysteine (ASC) and Se-propylselenocysteine (PSC), have been shown recently to be active in the chemoprevention of experimentally induced mammary carcinogenesis. Other than their potential as chemopreventive agents, little is known about the pharmacological properties of these compounds. In this article, we report on the in vitro effects of ASC and PSC on cell growth inhibition, apoptosis, and the induction of DNA damage. The effects of ASC and PSC were examined in two mouse mammary epithelial cell lines derived from mammary hyperplasias. These cell lines, designated TM2H and TM12, have mutant or wild-type p53, respectively. It was observed that ASC but not PSC reduced, in a concentration- and time-dependent manner, the number of adherent cells in culture, and this suppressive effect was more prominent in TM12 than in TM2H cells. ASC was also found to induce alkaline-labile DNA damage and the oxidation of pyrimidines, and it also increased the rate of apoptosis. These changes were not seen by exposure to PSC or the sulfur analog of ASC. However, additional data obtained from the intact rat mammary gland suggest that the loss of DNA integrity induced by ASC might not be manifest in vivo at doses of ASC that inhibit carcinogenesis.

In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention.

Previous research suggested that the beta-lyase-mediated production of a monomethylated selenium metabolite from Se-methylselenocysteine is a key step in cancer chemoprevention by this agent. In an attempt to affirm the concept, the present study was designed to evaluate the activity of methylseleninic acid, a compound that represents a simplified version of Se-methylselenocysteine without the amino acid moiety, thereby obviating the need for beta-lyase action. The in vitro experiments showed that methylseleninic acid was more potent than Se-methylselenocysteine in inhibiting cell accumulation and inducing apoptosis in TM12 (wild-type p53) and TM2H (nonfunctional p53) mouse mammary hyperplastic epithelial cells, and these effects were not attributable to DNA damage, as determined by the comet assay. In general, methylseleninic acid produced a more robust response at one-tenth the concentration of Se-methylselenocysteine. It is possible that these cell lines may have only a modest ability to generate a monomethylated selenium species from Se-methylselenocysteine via the beta-lyase enzyme. In contrast, methylseleninic acid already serves as a preformed active monomethylated metabolite, and this could be an underlying reason why methylseleninic acid acts more rapidly and exerts a more powerful effect than Se-methylselenocysteine in vitro. Interestingly, the distinction between these two compounds disappeared in vivo, where their cancer chemopreventive efficacies were found to be very similar to each other [in both methylnitrosourea and dimethylbenz(a)anthracene rat mammary tumor models]. The beta-lyase enzyme is present in many tissues; thus, animals have an ample capacity to metabolize Se-methylselenocysteine systemically. Therefore, Se-methylselenocysteine would be expected to behave like methylseleninic acid if beta-lyase is no longer a limiting factor. Taken together, the present in vitro and in vivo results provide strong evidence in support of our earlier hypothesis that a monomethylated selenium metabolite is important for cancer chemoprevention. Methylseleninic acid could be an excellent tool, especially for molecular mechanism studies in cell culture, and some of these attributes are discussed.

Plasma xanthophyll carotenoids correlate inversely with indices of oxidative DNA damage and lipid peroxidation.

Post hoc analysis of data obtained from a study designed to modulate oxidative damage by dietary intervention revealed consistently strong inverse correlations between plasma xanthophyll carotenoids and oxidative damage indices. Thirty-seven women participated in a 14-day dietary intervention that increased mean vegetable and fruit (VF) consumption to approximately 12 servings/day. An additional 10 subjects participated in an intervention that limited VF consumption to less than four servings per day. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) in DNA isolated from peripheral lymphocytes and 8-OHdG excreted in urine were measured as indices of oxidative DNA damage. Lipid peroxidation was assessed by measuring 8-epiprostaglandin F2alpha (8-EPG) in urine. Plasma levels of selected carotenoids were also determined, with the intention of using a-carotene as a biochemical index of VF consumption. Urinary 8-OHdG and 8-EPG were measured by ELISA, and plasma carotenoids were measured by high performance liquid chromatography. Lymphocyte 8-OHdG was measured by reverse phase high performance liquid chromatography with electrochemical detection. We observed that the structurally related xanthophyll carotenoids, lutein and beta-cryptoxanthin, which occur in dissimilar botanical families, were consistently inversely associated with these oxidative indices. Statistically significant inverse correlations were observed between plasma lutein and/or beta-cryptoxanthin levels and lymphocyte 8-OHdG and urinary 8-EPG. Moreover, an inverse correlation was observed between change in plasma xanthophylls and change in lymphocyte 8-OHdG concentration that occurred during the course of the study. These data lead us to hypothesize that lutein and beta-cryptoxanthin serve as markers for the antioxidant milieu provided by plants from which they are derived. Whether these carotenoids are directly responsible for the observed antioxidant phenomena merits further investigation.

A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human.

The injection of sexually immature female rats with 1-methyl-1-nitrosourea results in a rapid induction of premalignant and malignant mammary gland lesions within 35 days of carcinogen administration. This model affords the opportunity for investigators to study the process of mammary carcinogenesis over a very short latency and to investigate early events in this process. We have recently published on various aspects of this system including the histology of the lesions induced, the time frame of their occurrence, and their dependence on ovarian hormones for their maintenance and growth. In this report we present evidence that many aspects of the histopathology of mammary lesions in this model system are similar to those occurring in humans. We also discuss aspects of the human disease, which are not recapitulated in this model.

Selenium modulation of cell proliferation and cell cycle biomarkers in normal and premalignant cells of the rat mammary gland.

The present study was designed to assess the effect of Se-methylselenocysteine or triphenylselenonium chloride treatment on cell proliferation [bromodeoxyuridine (BrdUrd) labeling] and cell cycle biomarkers [proliferating cell nuclear antigen (PCNA), cyclin D1, and p27/Kip 1] in the intact mammary gland of rats. Immunohistochemical assays of the above end points were carried out in different morphological structures: (a) terminal end bud cells and alveolar cells of a maturing mammary gland undergoing active differentiation; and (b) premalignant mammary intraductal proliferations (IDPs) identified at 6 weeks after carcinogen dosing. Neither compound was found to affect BrdUrd labeling or the expression of cell cycle biomarkers in the normal terminal-end bud cells and alveolar cells. Se-methylselenocysteine reduced the total number of IDP lesions by approximately 60%. Interestingly, this was not accompanied by decreases in BrdUrd labeling or the proportion of IDP cells expressing PCNA and cyclin D1. An enhancement in the fraction of p27/Kip 1-positive IDP cells, however, was detected as a result of Se-methylselenocysteine treatment. Although triphenylselenonium chloride did not reduce the total number of IDPs, there were more of the smaller-sized lesions and fewer of the larger-sized lesions compared with those found in the control group. Triphenylselenonium chloride also significantly decreased the proportion of IDP cells incorporating the BrdUrd label or expressing PCNA and cyclin D1. The above findings suggest that early transformed cells are sensitive to selenium intervention, whereas normal proliferating cells are not. It is possible that Se-methylselenocysteine blocks carcinogenesis by a pathway that may not involve cell growth inhibition as a primary response; in contrast, triphenylselenonium chloride is likely to act by a cytostatic mechanism. The data also imply that selenium efficacy testing in intervention trials is possible with the use of biomarkers, provided that the appropriate biomarkers are matched with the selenium compound of interest and that the pathological characteristics of the cell population to be evaluated are taken into consideration.

Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.

Classification of premalignant and malignant lesions developing in the rat mammary gland after injection of sexually immature rats with 1-methyl-1-nitrosourea.

Premalignant and malignant stages of mammary carcinogenesis can be rapidly induced by injecting female rats i.p. with 1-methyl-1-nitrosourea (MNU)3 at 21 days of age. In this paper, the characteristics of this model are briefly reviewed and the histology of the lesions induced is presented and compared to those that occur in humans. Malignant mammary lesions induced in rats injected with MNU at 21 days of age are compared with the lesions that develop when MNU is administered to 50-day-old female rats.

Activity of Se-allylselenocysteine in the presence of methionine gamma-lyase on cell growth, DNA integrity, apoptosis, and cell-cycle regulatory molecules.

Se-allylselenocysteine (ASC) is effective in inhibiting mammary epithelial cell growth in vitro and mammary carcinogenesis in vivo, but its mechanism is unknown. We recently reported that ASC reduces cell growth in a dose- and time-dependent manner, induces a loss of DNA integrity, and increases apoptosis. However, the level of ASC required for growth inhibition in vitro is 10- to 20-fold higher than that required in vivo. One possible explanation for this difference is that the cells used in in vitro studies have limited lyase activity required to release the allyl Se moiety from selenocysteine, whereas animals have abundant lyase activity in tissues. In the present study, we found that methionine gamma-lyase (MGL) added to culture medium containing ASC produced biological effects with lower levels of ASC, comparable to the selenium levels in plasma achieved during in vivo chemoprevention. The combination of 2.5 microM ASC and MGL inhibited the growth of TM12 cells and increased apoptosis without loss of DNA integrity. Treatment of TM12 cells with ASC and MGL resulted in an elevation of the protein levels of p53, Cip1/p21, and Kip1/p27, concomitant with a decrease in cyclins D1 and E and modest reductions in cyclin-dependent kinase inhibitors 4 and 2. Cells treated with ASC and MGL also showed decreased phosphorylation of retinoblastoma tumor-suppressor protein. Taken together, these results suggest that a physiologically relevant concentration of ASC with MGL exerts an inhibitory effect on cell growth and that this effect is likely to involve modulation of signaling pathways that suppress the phosphorylation of retinoblastoma tumor-suppressor protein.

Rat models of premalignant breast disease.

While a number of agents have been shown to induce mammary carcinogenesis in the rat, premalignant stages of the disease have been best characterized in chemically-induced models, specifically those initiated by either 7,12 dimethylbenz[alpha]anthracene (DMBA) or 1-methyl-1-nitrosourea (MNU). In general, it appears that epithelial cells in mammary terminal end buds or terminal ductules are the targets of carcinogenic initiation, and that a series of morphologically identifiable steps are involved in the development of mammary carcinoma. The premalignant steps include ductal hyperplasia of the usual type and carcinoma in situ of the cribriform or comedo type; atypical ductal hyperplasia has not been reported. Thus the histogenesis of lesions occurring in chemically induced mammary carcinogenesis in the rat is similar to that observed in the human; although, the spectrum of lesions observed in the rat is limited. Opportunities to investigate the biological and molecular characteristics of premalignant breast disease in the rat are presented.

Effect of increased vegetable and fruit consumption on markers of oxidative cellular damage.

The goal of this study was to test the hypothesis that increased consumption of vegetables and fruit would reduce markers of oxidative cellular damage that can be assessed in blood or urine. Twenty-eight women participated in a 14 day dietary intervention. The primary end-points assessed were: 8-hydroxydeoxyguanosine (8-OHdG) in DNA isolated from peripheral lymphocytes, determined by HPLC with electrochemical detection; 8-OHdG excreted in urine, measured by ELISA; malondialdehyde (MDA) in urine, measured by fluorimetric detection following derivatization with thiobarituric acid and separation via HPLC; urinary 8-isoprostane F-2alpha (8-EPG) detected by ELISA. Pre- and post-intervention plasma levels of selected carotenoids were determined by HPLC. Subjects were free living and consumed a completely defined recipe-based diet that increased their average daily consumption of vegetables and fruit from 5.8 servings at baseline to 12.0 servings throughout the intervention. Overall, the level of 8-OHdG in DNA isolated from lymphocytes and in urine and the level of 8-EPG in urine were reduced by the intervention, whereas urine concentrations of MDA were minimally affected. The reduction in lymphocyte 8-OHdG was greater in magnitude (32 versus 5%) in individuals with lower average pre-intervention levels of plasma alpha-carotene (56 ng/ml) than in individuals with higher average pre-intervention plasma levels of alpha-carotene (148 ng/ml). The results of this study indicate that consumption of a diet that significantly increased vegetable and fruit intake from a diverse number of botanical families resulted in significant reductions in markers of oxidative cellular damage to DNA and lipids.

Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats.

Conjugated linoleic acid (CLA) is a potent cancer preventive agent in animal models. To date, all of the in vivo work with CLA has been done with a commercial free fatty acid preparation containing a mixture of c9,t11-, t10,c12- and c11,t13-isomers, although CLA in food is predominantly (80-90%) the c9,t11-isomer present in triacylglycerols. The objective of this study was to determine whether a high CLA butter fat has biological activities similar to those of the mixture of free fatty acid CLA isomers. The following four different endpoints were evaluated in rat mammary gland: 1) digitized image analysis of epithelial mass in mammary whole mount; 2) terminal end bud (TEB) density; 3) proliferative activity of TEB cells as determined by proliferating cell nuclear antigen immunohistochemistry; and 4) mammary cancer prevention bioassay in the methylnitrosourea model. It should be noted that TEB cells are the target cells for mammary chemical carcinogenesis. Feeding butter fat CLA to rats during the time of pubescent mammary gland development reduced mammary epithelial mass by 22%, decreased the size of the TEB population by 30%, suppressed the proliferation of TEB cells by 30% and inhibited mammary tumor yield by 53% (P < 0.05). Furthermore, all of the above variables responded with the same magnitude of change to both butter fat CLA and the mixture of CLA isomers at the level of CLA (0.8%) present in the diet. Interestingly, there appeared to be some selectivity in the uptake or incorporation of c9,t11-CLA over t10,c12-CLA in the tissues of rats given the mixture of CLA isomers. Rats consuming the CLA-enriched butter fat also consistently accumulated more total CLA in the mammary gland and other tissues (four- to sixfold increases) compared with those consuming free fatty acid CLA (threefold increases) at the same dietary level of intake. We hypothesize that the availability of vaccenic acid (t11-18:1) in butter fat may serve as the precursor for the endogenous synthesis of CLA via the Delta9-desaturase reaction. Further studies will be conducted to investigate other attributes of this novel dairy product.

Effect of energy restriction on tissue size regulation during chemically induced mammary carcinogenesis.

Energy restriction (ER) has documented beneficial effects on numerous diseases including cancer, yet the mechanism(s) that accounts for these effects is unknown. Experiments were designed to determine the effect of ER: (i) on the growth and development of the mammary gland; (ii) on the growth of carcinomas induced in the mammary gland by treatment with 1-methyl-1-nitrosourea (MNU); (iii) on rates of cell proliferation and apoptosis in pre-malignant and malignant mammary lesions. Mammary carcinogenesis was induced in female Sprague-Dawley rats by the i.p. administration of MNU (50 mg MNU/kg body wt) at 21 days of age. Rats were randomized to one of four dietary treatment groups: ad libitum fed or restriction of calorie intake to 90, 80 or 60% of ad libitum intake. ER reduced the ductal extension of the mammary gland into the fat pad in proportion to its effect on growth measured as body weight, however, the reduction in ductal branching, breast density and carcinoma volume by ER was greater than its effect on body weight. An animal's breast density was predictive of its carcinogenic response, irrespective of the level of ER imposed. While ER inhibited cell proliferation and induced apoptosis in pre-malignant and malignant mammary gland lesions, the magnitude of these effects make it unlikely that they fully account for the protective effects of ER against mammary carcinogenesis.

Effect of energy restriction on the expression of cyclin D1 and p27 during premalignant and malignant stages of chemically induced mammary carcinogenesis.

The restriction of energy intake has a profound inhibitory effect on carcinogenesis, yet the mechanism or mechanisms that account for this effect are unknown. In this experiment, the hypothesis tested was that energy restriction upregulates the expression of p27/kip1, a gene product associated with cell-cycle growth arrest, while downregulating cyclin D1, a protein that combines with cyclin-dependent kinases to promote phosphorylation of retinoblastoma protein and the progression of cells through the cell cycle. We studied levels of these proteins in uninvolved mammary epithelial cells and in mammary intraductal proliferations, ductal carcinomas in situ, and adenocarcinomas induced in response to administration of 1-methyl-1-nitrosourea in animals fed either ad libitum or 90%, 80%, or 60% of ad libitum intake. Protein levels were evaluated immunohistochemically by using computer-assisted image analysis to quantify differences in protein expression among treatment groups. The expression of p27 increased and the expression of cyclin D1 decreased dose-dependently in response to energy restriction. The effect was greater on p27 than on cyclin D1. The hypothesis proposed is that energy restriction inhibits carcinogenesis by arresting cell-cycle progression by regulating p27/kip1.

Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.

Methylselenocysteine (MSC), an organic selenium compound is an effective chemopreventive agent against mammary cell growth both in vivo and in vitro but its mechanism of action is still not understood. We have previously demonstrated that MSC is able to inhibit growth in a synchronized TM6 mouse mammary epithelial tumor cell line at 16 h time point followed by apoptosis at 48 h. The decrease in cdk2 kinase activity was coincident with prolonged arrest of cells in S-phase. The present set of experiments showed that cdk2 phosphorylation was reduced by 72% in the MSC-treated cells at 16 h time point. Expression for gadd34, 45 and 153 was elevated 2.5 to 7 fold following MSC treatment only after 16 h time point. In order to investigate a possible upstream target for MSC, we analyzed protein kinase C (PKC) in this model. Total PKC activity was reduced in TM6 cells by MSC (50 microM) within 30 min of treatment, both in cytosolic (55.4 and 77.6%) and membrane (35.2 and 34.1%) fractions for calcium-dependent and independent PKCs, respectively. PMA significantly elevated the PKC activity in membrane fraction (P < 0.01) and MSC inhibited this activation by more than 57%. The effect of MSC was selenium specific as selenomethionine and sulfurmethyl-L-cysteine (SMC) did not alter PKC activity either in cytosolic or membrane fraction. Immunoblot analysis showed that PKC-alpha was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-delta was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-e (in cytosolic and membrane fractions) and PKC-zeta (cytosolic fractions). The data presented herein suggest that PKC is a potential upstream target for MSC that may trigger one or all of the downstream effects; i.e. the decrease of cdk2 kinase activity, decreased DNA synthesis, elevation of gadd gene expression and finally apoptosis.