Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate.

Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor alpha mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor alpha positive, MCF-7 cells increased peroxisome proliferator-activated receptor alpha mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4h of treatment. Peroxisome proliferator-activated receptor alpha modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor alpha negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors.

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines.

The plasma membrane Ca(2+) ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca(2+) plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca(2+) regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.

Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin.

Peroxisome proliferator-activated receptor beta (PPARbeta) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor, although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARbeta with colon cancer and increased mRNA levels of PPARbeta in colon tumours we sought in this study to examine the expression of PPARbeta in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARbeta mRNA levels in MCF-7, MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARbeta compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARbeta mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARbeta was inducible upon exposure to the PPARbeta ligand bezafibrate. Our results suggest that unlike colon cancer, PPARbeta overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARbeta mRNA expression and the ability of PPARbeta in the MCF-7 cells to respond to ligand indicates that PPARbeta may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists.

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader.

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.

Antisense-mediated Inhibition of the plasma membrane calcium-ATPase suppresses proliferation of MCF-7 cells.

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Effects of peroxisome proliferator-activated receptor gamma ligands ciglitazone and 15-deoxy-delta 12,14-prostaglandin J2 on rat cultured cerebellar granule neuronal viability.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARgamma ligands. Comparison of these studies is complicated by the use of different PPARgamma ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARgamma ligand ciglitazone with an endogenous PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2) (15-deoxy PGJ(2)), on inherent neurotoxicity and neuroprotection using a reduction in extracellular KCl in rat cultured cerebellar granule neurons (CGN). We also assessed the effects of these ligands on c-Jun protein expression, which is up-regulated on induction of low-KCl-mediated neuronal apoptosis as well as being associated with PPAR in other cell types. We showed that PPARgamma mRNA is expressed in CGN cultures and observed ciglitazone- and 15-deoxy PGJ(2)-mediated inherent neurotoxicity that was concentration and time dependent. c-Jun was only modestly increased in the presence of ciglitazone but was markedly up-regulated by 15-deoxy PGJ(2) after 12 hr. Treatment of CGN cultures with ciglitazone simultaneous with KCl withdrawal resulted in a modest, time-dependent neuroprotection. Such neuroprotection after KCl withdrawal was not observed with 15-deoxy PGJ(2). Despite the absence of neuroprotection, 15-deoxy PGJ(2) markedly inhibited the early up-regulation of c-Jun during KCl withdrawal. These studies suggest that ciglitazone and 15-deoxy PGJ(2) have markedly different effects on inherent and low-KCl-induced toxicity and c-Jun expression in CGN, indicating potential non-PPARgamma mechanisms.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231.

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARalpha activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.