Comparative effects of antioxidants on enzymes involved in glutathione metabolism

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, γ-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, γ-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

Hydrocortisone and progesterone regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1–10 μg/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01–1 μg/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 μg/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells. © 1995 Wiley-Liss, Inc.     

X-chromosome inactivation in the Wiskott-Aldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.     

Cell movements during gastrulation: snail dependent and independent pathways

The morphogenetic process of gastrulation requires multiple inputs and intricate coordination. Genetic analyses demonstrate critical roles of vertebrate and invertebrate Snail proteins in this process. Together with other regulatory molecules including Wnt and BMP, the Snail pathways specify cell fate and reorganize cellular machineries to coordinate morphological changes and cell movements during gastrulation.     

PTEN Loss Promotes Mitochondrially Dependent Type II Fas-Induced Apoptosis via PEA-15

Two distinct biochemical signals are delivered by the CD95/Fas death receptor. The molecular basis for the differential mitochondrially independent (type I) and mitochondrially dependent (type II) Fas apoptosis pathways is unknown. By analyzing 24 Fas-sensitive tumor lines, we now demonstrate that expression/activity of the PTEN tumor suppressor strongly correlates with the distinct Fas signals. PTEN loss-of-function and gain-of-function studies demonstrate the ability to interconvert between type I and type II Fas pathways. Importantly, from analyses of Bcl-2 transgenic Pten^+/− mice, Pten haploinsufficiency converts Fas-induced apoptosis from a Bcl-2-independent to a Bcl-2-sensitive response in primary thymocytes and activated T lymphocytes. We further show that PTEN influences Fas signaling, at least in part, by regulating PEA-15 phosphorylation and activity that, in turn, regulate the ability of Bcl-2 to suppress Fas-induced apoptosis. Thus, PTEN is a key molecular rheostat that determines whether a cell dies by a mitochondrially independent type I versus a mitochondrially dependent type II apoptotic pathway upon Fas stimulation.Two types of Fas apoptotic signaling pathways, designated the type I and type II pathways, occur in distinct classes of cells (2). Biochemically, type I and type II cells differ primarily in the amounts of FADD and caspase-8 recruited to the Fas receptor, in the kinetics of caspase cascade activation, and in their relative dependence on the mitochondrial intrinsic arm of the Fas apoptotic pathway in the execution of cell death (34). Fas receptor aggregation leads to the recruitment of the adaptor protein FADD and the initiator caspase-8 and -10, forming the death-inducing signaling complex (DISC) and resulting in autoproteolytic activation of these caspases. In type I cells, a sufficient amount of caspase-8 is processed to directly activate the effector caspase-3 and to execute apoptosis. While the intrinsic mitochondrial apoptotic pathway is also activated in type I cells, the relative contribution of this branch to apoptosis induction is diminished by the potent action of the direct pathway. In contrast to type I cells and despite similar expression of cell surface Fas, type II cells form a weak DISC and exhibit delayed kinetics of caspase-8 and -3 activation. Due to the paucity of FADD recruitment and caspase-8 processing at the DISC in type II cells, the direct activation of caspase-3 is attenuated, resulting in the increased dependence of type II cells on the mitochondrial amplification loop activated by the proapoptotic Bcl-2 member Bid in order to execute apoptosis. Hence, type I cells undergo Fas-mediated apoptosis in a mitochondrially independent manner, whereas type II cells have increased dependence on the intrinsic mitochondrial pathway to induce apoptosis.Despite an intensive search, the identity of the signaling protein(s) that determines whether a cell dies by type I versus type II Fas-induced apoptosis has remained elusive (28). By virtue of their ability to regulate Fas signaling in various tissue types, a plethora of signaling proteins, including death receptor signaling proteins such as DAXX, FAP-1, FAF1, FLASH, RIP, and FLIP, apoptosis regulatory proteins such as IAP family members, Bcl-2-related proteins, and signaling proteins such as PP2A, CaMKII, PEA-15, galectin-3, PTEN, PI3K, and PKB, among others, have been implicated as potential candidates (8-11, 13-16, 21, 28, 42, 46).In search of the signaling pathway(s) that is differentially activated in type I and type II cells, we performed a Kinetworks phosphosite screen (KPSS1.3), which simultaneously detects the presence and relative quantities of 34 critical protein phosphorylation sites, and found that the serine/threonine protein kinase B (PKB; also known as Akt) was highly phosphorylated in prototypic type II Jurkat but not type I H9 cells (Kinexus, Vancouver, BC) (data not shown). Furthermore, we noted that both of the prototypic type II cell lines, i.e., Jurkat and CEM, are known to be deficient in the PTEN tumor suppressor (33). Therefore, we hypothesized that PTEN may be an important regulator of the differential Fas signaling pathways in type I and type II cells.The PTEN tumor suppressor gene is among the most commonly mutated genes in a broad range of human malignancies. PTEN is an important negative regulator of cell growth and survival. Among other functions, PTEN is a phosphatidylinositol 3′-phosphatase that specifically downmodulates the levels of phosphoinositide second messengers such as phosphatidylinositol(3,4,5)-trisphosphate, thereby antagonizing the action of phosphatidylinositol 3-kinase (PI3K). Loss of PTEN function results in increased membrane phosphatidylinositol(3,4,5)-trisphosphate levels and constitutive activation of its downstream effectors, such as PKB, leading to enhanced cellular metabolism, growth, and survival (26).In this study, we investigated whether the PI3K/PTEN pathway may be important in regulating Fas-induced apoptosis in type I and type II cells. Indeed, we found a robust correlation between PTEN expression and type I/II Fas-induced apoptosis in a wide variety of cancers. Furthermore, through PTEN gain-of-function and loss-of-function approaches, we demonstrated the ability of the PI3K/PTEN pathway to promote interconversion between the mitochondrially independent type I and mitochondrially dependent type II Fas pathways. Significantly, we found that PTEN haploinsufficiency promotes Bcl-2 sensitivity of Fas-induced apoptosis of primary thymocytes and activation-induced cell death of T lymphocytes. Furthermore, Bcl-2 sensitivity of Fas-induced apoptosis was found to be regulated by PEA-15, in a phosphorylation-dependent manner, and PEA-15 phosphorylation is mediated by the PTEN/PI3K pathway. Thus, our data indicate that the PTEN/PI3K pathway modulates the dependency of cells on the mitochondrial amplification loop to mediate Fas-induced apoptosis and determines whether a cell dies by a type I or type II Fas pathway, in part through regulating PEA-15 activity.     

Constitutively Nuclear FOXO3a Localization Predicts Poor Survival and Promotes Akt Phosphorylation in Breast Cancer

Background

The PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis.

Methodology/Principal Findings

Examination of FOXO3a and phosphorylated-Akt (P-Akt) expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.052), poor prognosis (p = 0.014), and P-Akt expression in invasive ductal carcinoma. Using tamoxifen and doxorubicin-sensitive and -resistant breast cancer cell lines as models, we found that doxorubicin- but not tamoxifen-resistance is associated with nuclear accumulation of FOXO3a, consistent with the finding that sustained nuclear FOXO3a is associated with poor prognosis. We also established that doxorubicin treatment induces proliferation arrest and FOXO3a nuclear relocation in sensitive breast cancer cells. Induction of FOXO3a activity in doxorubicin-sensitive MCF-7 cells was sufficient to promote Akt phosphorylation and arrest cell proliferation. Conversely, knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity. Using MDA-MB-231 cells, in which FOXO3a activity can be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction up-regulates PI3K-Akt activity and enhanced doxorubicin resistance. However FOXO3a induction has little effect on cell proliferation, indicating that FOXO3a or its downstream activity is deregulated in the cytotoxic drug resistant breast cancer cells. Thus, our results suggest that sustained FOXO3a activation can enhance hyperactivation of the PI3K/Akt pathway.

Conclusions/Significance

Together these data suggest that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signaling axis. In these breast cancers activated Akt fails to inactivate and re-localize FOXO3a to the cytoplasm, and nuclear-targeted FOXO3a does not induce cell death or cell cycle arrest. As such, sustained nuclear FOXO3a expression in breast cancer may culminate in cancer progression and the development of an aggressive phenotype similar to that observed in cytotoxic chemotherapy resistant breast cancer cell models.     

Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells

Introduction  

This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation.