Early de novo gene expression is required for 15-deoxy-Delta 12,14-prostaglandin J2-induced apoptosis in breast cancer cells.

Cyclopentenone prostaglandin derivatives of arachidonic acid are potent inducers of apoptosis in a variety of cancer cell types. Several investigators have shown that the terminal derivative of prostaglandin J(2) (PGJ(2)) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), but 15dPGJ(2) effects can be mediated by PPARgamma-dependent and PPARgamma-independent mechanisms. Here we report that 15dPGJ(2) regulates early gene expression critical to apoptosis. Specifically, 15dPGJ(2) induces potent and irreversible S phase arrest that is correlated with expression of genes critical to cell cycle arrest and apoptosis, including the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Inhibition of RNA or protein synthesis abrogates apoptosis induced by 15dPGJ(2) in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor-alpha or CD95/Fas ligand. Additionally, 15dPGJ(2) induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ(2)-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

Magnitude of peroxisome proliferator-activated receptor-gamma activation is associated with important and seemingly opposite biological responses in breast cancer cells.

BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARgamma leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARgamma agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors. METHODS: We studied the effects of low-, moderate-, and high-dose treatment of the PPARgamma ligands 15-deoxy-delta1214 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231. RESULTS: The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARgamma activation. For example, low concentrations of 15dPGJ2 (<2.5 microM) and TGZ (<5 microM) increased cellular proliferation, but concentrations of 15dPGJ2 > or = 10 microM and of TGZ at 100 microM blocked cell growth. TGZ (100 microM) slowed cell cycle progression, and 15dPGJ2 (10 microM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 microM 15dPGJ2 or 5 to 100 microM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARgamma ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis. CONCLUSIONS: PPARgamma activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARgamma activation. These data may explain, at least in part, some of the discordant results previously reported.

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Influence of coenzyme A-independent transacylase and cyclooxygenase inhibitors on the proliferation of breast cancer cells.

Recent studies have demonstrated that arachidonic acid (AA) may serve as an important signal that blocks cell proliferation of certain neoplastic cells. The current study was conducted to determine whether disruption of AA homeostasis influences breast cancer cell proliferation and death. Initial experiments revealed that inhibition of AA remodeling through membrane phospholipids by inhibitors of the enzyme, coenzyme A-independent transacylase (CoA-IT), attenuates the proliferation of the estrogen receptor-negative, MDA-MB-231, and estrogen receptor-positive, MCF-7 breast cancer cell lines. This growth inhibition was accompanied by a marked accumulation of AA in both free fatty acid and triglyceride forms, a marker of intracellular AA stress within mammalian cells. Cell cycle synchronization experiments revealed that the CoA-IT inhibitor, SB-98625, blocked MDA-MB-231 cell replication in early to mid G1 phase. Time-lapse video microscopy, used to observe the changes in cell morphology associated with apoptosis, indicated that SB-98625 treatment induced early rounding and occasional blebbing but not late apoptotic events, blistering, and lysis. The cyclooxygenase inhibitors, NS-398 and indomethacin, were found to be less potent blockers of cell proliferation and poor inducers of cellular AA accumulation than CoA-IT inhibitors in these breast cancer cell lines. Finally, AA provided exogenously blocked the proliferation of MCF-7 cells, and this effect could be attenuated in MCF-7 cells overexpressing the glutathione peroxidase gene, GSHPx-1. Taken together, these experiments suggest that disruption of AA remodeling in a manner that increases intracellular AA may represent a novel therapeutic strategy to reduce cancer cell proliferation and that an oxidized AA metabolite is likely to mediate this effect.

The effects of ethanol and acetaldehyde on the metabolism of prostaglandin E2 and leukotriene B4 in isolated rat hepatocytes.

The effects of ethanol and acetaldehyde on the metabolism of leukotriene B4 (LTB4) and PGE2 were investigated in isolated cultures of rat hepatocytes. LTB4 undergoes initial cytochrome P450-dependent omega-oxidation leading to the principal metabolites 20-hydroxy-LTB4, 20-carboxy-LTB4 and the omega/beta-oxidation product 18-carboxy-LTB4. The addition of low concentrations of ethanol (25 mM) dramatically changes the relative amounts of these metabolite products by inhibiting the alcohol dehydrogenase-mediated oxidation of 20-hydroxy-LTB4. Addition of acetaldehyde to the incubation, up to 1 mM, had no significant effect on overall metabolism or distribution of metabolites. Above 1 mM acetaldehyde, beta-oxidation of LTB4 was inhibited. Thus the effect of ethanol on the metabolism of LTB4 appears to be due to ethanol itself and not to secondary effects from the metabolic transformation of ethanol to acetaldehyde in the cells. PGE2 is metabolized in isolated rat hepatocytes to produce chain-shortened products of beta-oxidation characterized as dinor-PGE1, dinor-PGE2, tetranor-PGE1, tauro-dinor-PGE1 and tauro-dinor-PGE2. Low concentrations of ethanol (25 mM) were found to increase the relative concentration of dinor-PGE1 in the metabolic distribution, with a corresponding decrease in concentration of tetranor-PGE1. The amount of dinor-PGE2 that was produced remained relatively unchanged in response to increasing concentrations of ethanol. Acetaldehyde concentrations from 0.1 mM to 1 mM did not affect metabolite distribution or the overall magnitude of PGE2 metabolism. Concentrations of acetaldehyde higher than 1 mM decreased all beta-oxidation metabolites. Ethanol, at physiologically relevant concentrations, could alter eicosanoid metabolism in the liver by inhibiting LTB4 metabolism and altering that of PGE2.

Effect of faecal extract on the growth of Salmonella enteritidis in artificially contaminated hens' eggs.

1. Salmonella enteritidis PT 4 grew in eggs stored at 25 degrees C, but not at 10 degrees C. 2. The incidence of generalised infection of the egg contents (greater than 10(6) salmonellas/ml) was greater in eggs inoculated with cells suspended in faecal extract compared to those with cells in Ringer's solution. 3. The removal of most of the iron did not decrease the growth-promoting effect of the faecal extract.

Growth of Salmonella enteritidis in artificially contaminated hens' shell eggs.

The effect of some factors on the growth of Salmonella enteritidis phage type 4 in artificially contaminated shell eggs was investigated. Salmonella enteritidis was found to be resistant to the antimicrobial properties of the albumen. Growth occurred on storage at 25 degrees C but not at 4 or 10 degrees C. The rate and extent of infection was influenced by the size of inoculum, the site of contamination relative to yolk movement, and the presence of iron in the inoculum.

Congenital nuclear cataracts in cattle.

A description is given of the investigation into two outbreaks of congenital cataract in dairy herds. There is a description of the clinical signs, the incidence, the period of time over which the cases occurred and of the seasonal distribution of the births. The type of herd management is descirbed and a hereditary factor is not considered to occur. The possibility of the involvement of other toxic and environmental agents is discussed. The macroscopic and microscopical findings are described; the relation to the early period of lens development of the bovine fetus suggested.