Kathleen A. O'Leary

mdm2 Is Critical for Inhibition of p53 during Lymphopoiesis and the Response to Ionizing Irradiation

ABSTRACT The function of the p53 tumor suppressor protein must be highly regulated because p53 can cause cell death and prevent tumorigenesis. In cultured cells, the p90MDM2 protein blocks the transcriptional activation domain of p53 and also stimulates the degradation of p53. Here we provide the first conclusive demonstration that p90MDM2 constitutively regulates p53 activity in homeostatic tissues. Mice with a hypomorphic allele of mdm2 revealed a heretofore unknown role for mdm2 in lymphopoiesis and epithelial cell survival. Phenotypic analyses revealed that both the transcriptional activation and apoptotic functions of p53 were increased in these mice. However, the level of p53 protein was not coordinately increased, suggesting that p90MDM2 can inhibit the transcriptional activation and apoptotic functions of p53 in a manner independent of degradation. Cre-mediated deletion of mdm2 caused a greater accumulation of p53, demonstrating that p90MDM2 constitutively regulates both the activity and the level of p53 in homeostatic tissues. The observation that only a subset of tissues with activated p53 underwent apoptosis indicates that factors other than p90MDM2 determine the physiological consequences of p53 activation. Furthermore, reduction of mdm2 in vivo resulted in radiosensitivity, highlighting the importance of mdm2 as a potential target for adjuvant cancer therapies.

Purification of three cytosolic glutathione S-transferases from adult Schistosoma mansoni☆

The cytosolic fraction of adult Schistosoma mansoni contains glutathione S-transferase (EC 2.5.1.18) activity, determined with the prototype substrate 1-chloro-2,4-dinitrobenzene, that is 5- to 50-fold greater than that found in other metazoan parasites. A survey of several model substrates revealed that enzymes in male and female schistosomes have distinct but overlapping substrate specificities. Four forms of glutathione S-transferase were detected, three of which, SmGST-1, SmGST-2, and SmGST-3, were purified to apparent homogeneity by glutathione affinity chromatography and HPLC chromatofocusing. The purified enzymes displayed very similar catalytic and physicochemical properties. They could be distinguished by differences in activity with ethacrynic acid and trans-4-phenyl-3-buten-2-one, but not with aryl halide substrates. The isoelectric points of SmGST-1, SmGST-2, and SmGST-3 were estimated to be 7.2, 7.1, 6.9, respectively. A polyclonal antiserum to SmGST-3 cross-reacted with the other two forms, but not with other soluble schistosome proteins. Each of the purified enzymes displayed an apparent subunit molecular weight of 28,500 by polyacrylamide gel electrophoresis under denaturing conditions. Gel filtration chromatography yielded a molecular weight of 30,800 for the catalytically active form of the enzyme. Unlike all known glutathione S-transferases, the three enzyme forms purified from S. mansoni appear to be catalytically active monomeric proteins.

IMMUNOCYTOCHEMICAL LOCALIZATION OF THE MAJOR GLUTATHIONE S-TRANSFERASES IN ADULT SCHISTOSOMA MANSONI

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.

Schistosoma mansoni : single-step purification and characterization of glutathione S-transferase isoenzyme 4

A soluble glutathione S-transferase isoenzyme, designated SmGST-4 was purified to apparent homogeneity in a single step from the cytosol of adult Schistosoma mansoni by selective elution of the enzyme from a glutathione-agarose affinity column using glutathione disulfide. SmGST-4, which comprised about 5% of the bound glutathione S-transferase activity, could be distinguished from the previously characterized glutathione S-transferase isoenzyme family (SmGST-1/2/3), by its unique chromatographic behavior, lower subunit M(r) (26,000), differences in substrate specificity and inhibitor sensitivity, and a lack of reactivity with antiserum to SmGST-3. The purified isoenzyme catalyzed the conjugation of several model xenobiotics including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and trans-4-phenyl-3-buten-2-one. Like the SmGST-1/2/3 isoenzyme family, SmGST-4 failed to catalyze the conjugation of a model epoxide substrate, 1,2-epoxy-3-(p-nitrophenoxy)propane. Because glutathione S-transferases from other organisms play a role in protecting cells against the toxic products of lipid peroxidation, SmGST-4 and the members of the SmGST-1/2/3 isoenzyme family were tested for their capacity to reduce cumene hydroperoxide and to catalyze the conjugation of 4-hydroxyalk-2-enals. Although all four isoenzymes catalyzed both reactions, the specific activity of SmGST-1, SmGST-2, and SmGST-3 toward cumene hydroperoxide was at least 10-fold greater than that of SmGST-4. In contrast, the latter more effectively conjugated a homologous series of 4-hydroxyalk-2-enal isomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Schistosoma mansoni : glutathione S-transferase-catalyzed detoxication of dichlorvos

Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathione-dependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, S-methylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione S-transferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH S-transferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate.

Mdm2 Regulates p53 Independently of p19ARF in Homeostatic Tissues

ABSTRACT Tumor suppressor proteins must be exquisitely regulated since they can induce cell death while preventing cancer. For example, the p19ARF tumor suppressor (p14ARF in humans) appears to stimulate the apoptotic function of the p53 tumor suppressor to prevent lymphomagenesis and carcinogenesis induced by oncogene overexpression. Here we present a genetic approach to defining the role of p19ARF in regulating the apoptotic function of p53 in highly proliferating, homeostatic tissues. In contrast to our expectation, p19ARF did not activate the apoptotic function of p53 in lymphocytes or epithelial cells. These results demonstrate that the mechanisms that control p53 function during homeostasis differ from those that are critical for tumor suppression. Moreover, the Mdm2/p53/p19ARF pathway appears to exist only under very restricted conditions.

Social isolation reduces mammary development, tumor incidence, and expression of epigenetic regulators in wild-type and p53-heterozygotic mice.

Chronic stress is associated with more rapid tumor progression, and recent evidence suggests that stress may contribute to social and ethnic disparities in the incidence and mortality of breast cancer. We evaluated the p53+/− FVB/N mouse as a model to investigate effects of chronic social stress on mammary gland development, gene expression, and tumorigenesis. We individually housed (IH) wild-type and p53+/− female FVB/N mice, starting at weaning. At 14 weeks of age, both wild-type and p53+/− IH mice showed strikingly reduced mammary development compared with group-housed (GH) controls, with IH mice having significantly fewer preterminal end buds. This morphologic difference was not reflected in levels of mammary transcripts for estrogen receptor-α or progestin receptor. However, IH increased levels of mRNA for the kisspeptin receptor in the medial preoptic area of the hypothalamus, associated with reduced duration of estrous cycles. Furthermore, IH altered mammary transcripts of genes associated with DNA methylation; transcripts for methyl-binding protein 2 and DNA methyltransferase 3b (DNMT3b), but not DNMT1 and DNMT3a, were reduced in IH compared with GH females. Interestingly, the glands of p53+/− females showed reduced expression of all these mediators compared with wild-type females. However, contrary to our initial hypothesis, IH did not increase mammary tumorigenesis. Rather, p53+/− GH females developed significantly more mammary tumors than IH mice. Together, these data suggest that social isolation initiated at puberty might confound studies of tumorigenesis by altering mammary development in mouse models. Cancer Prev Res; 3(5); 620–9. ©2010 AACR.

Prolactin Activates ERα in the Absence of Ligand in Female Mammary Development and Carcinogenesis in Vivo

Resistance of estrogen receptor positive (ERα+) breast cancers to antiestrogens is a major factor in the mortality of this disease. Although activation of ERα in the absence of ligand is hypothesized to contribute to this resistance, the potency of this mechanism in vivo is not clear. Epidemiologic studies have strongly linked prolactin (PRL) to both development of ERα+ breast cancer and resistance to endocrine therapies. Here we employed genetically modified mouse models to examine the ability of PRL and cross talk with TGFα to activate ERα, using a mutated ERα, ERα(G525L), which is refractory to endogenous estrogens. We demonstrate that PRL promotes pubertal ERα-dependent mammary ductal elongation and gene expression in the absence of estrogen, which are abrogated by the antiestrogen, ICI 182,780 (ICI). PRL and TGFα together reduce sensitivity to estrogen, and 30% of their combined stimulation of ductal proliferation is inhibited by ICI, implicating ligand-independent activation of ERα as a component of their interaction. However, PRL/TGFα-induced heterogeneous ERα+ tumors developed more rapidly in the presence of ICI and contained altered transcripts for surface markers associated with epithelial subpopulations and increased signal transducer and activator of transcription 5b expression. Together, these data support strong interactions between PRL and estrogen on multiple levels. Ligand-independent activation of ERα suggests that PRL may contribute to resistance to antiestrogen therapies. However, these studies also underscore ERα-mediated moderation of tumor phenotype. In light of the high expression of PRL receptors in ERα+ cancers, understanding the actions of PRL and cross talk with other oncogenic factors and ERα itself has important implications for therapeutic strategies.

Modeling prolactin actions in breast cancer in vivo: insights from the NRL-PRL mouse.

Elevated exposure to prolactin (PRL) is epidemiologically associated with an increased risk of aggressive ER+ breast cancer. To understand the underlying mechanisms and crosstalk with other oncogenic factors, we developed the NRL-PRL mouse. In this model, mammary expression of a rat prolactin transgene raises local exposure to PRL without altering estrous cycling. Nulliparous females develop metastatic, histotypically diverse mammary carcinomas independent from ovarian steroids, and most are ER+. These characteristics resemble the human clinical disease, facilitating study of tumorigenesis, and identification of novel preventive and therapeutic approaches.

Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53−/− carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53−/− carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial–mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.