N. A. Holman

Peroxisome proliferator–activated receptor α in the human breast cancer cell lines MCF‐7 and MDA‐MB‐231

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα 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, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.

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

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα 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, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.

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 β 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.

Effects of peroxisome proliferator-activated receptor γ ligands ciglitazone and 15-deoxy-Δ12,14-prostaglandin J2 on rat cultured cerebellar granule neuronal viability

Peroxisome proliferator‐activated receptor γ (PPARγ) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARγ ligands. Comparison of these studies is complicated by the use of different PPARγ ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARγ ligand ciglitazone with an endogenous PPARγ ligand, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15‐deoxy PGJ2), 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 PPARγ mRNA is expressed in CGN cultures and observed ciglitazone‐ and 15‐deoxy PGJ2‐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 PGJ2 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 PGJ2. Despite the absence of neuroprotection, 15‐deoxy PGJ2 markedly inhibited the early up‐regulation of c‐Jun during KCl withdrawal. These studies suggest that ciglitazone and 15‐deoxy PGJ2 have markedly different effects on inherent and low‐KCl‐induced toxicity and c‐Jun expression in CGN, indicating potential non‐PPARγ mechanisms.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca(2+) pump is a key regulator of cytosolic free Ca(2+). Recent studies have demonstrated the dynamic expression of the plasma membrane Ca(2+) pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca(2+) ATPase (PMCA1) isoform of the plasma membrane Ca(2+) pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.

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

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα 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, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.

Effects of peroxisome proliferator-activated receptor gamma ligands ciglitazone and 15-deoxy-delta 12,14-prostaglandin J2 on rat cultured cerebellar granule neuronal viability Steven A. Smith, Gregory R. Monteith, Nicola A. Holman, Jodie A. Robinson, Fiona J. May, Sarah J. Roberts-Thomson

Peroxisome proliferator‐activated receptor γ (PPARγ) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARγ ligands. Comparison of these studies is complicated by the use of different PPARγ ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARγ ligand ciglitazone with an endogenous PPARγ ligand, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15‐deoxy PGJ2), 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 PPARγ mRNA is expressed in CGN cultures and observed ciglitazone‐ and 15‐deoxy PGJ2‐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 PGJ2 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 PGJ2. Despite the absence of neuroprotection, 15‐deoxy PGJ2 markedly inhibited the early up‐regulation of c‐Jun during KCl withdrawal. These studies suggest that ciglitazone and 15‐deoxy PGJ2 have markedly different effects on inherent and low‐KCl‐induced toxicity and c‐Jun expression in CGN, indicating potential non‐PPARγ mechanisms.

Effects of peroxisome proliferator-activated receptor gamma ligands ciglitazone and 15-deoxy-delta 12,14-prostaglandin J2 on rat cultured cerebellar granule neuronal viability Steven A. Smith, Gregory R. Monteith, Nicola A. Holman, Jodie A. Robinson, Fion

Peroxisome proliferator‐activated receptor γ (PPARγ) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARγ ligands. Comparison of these studies is complicated by the use of different PPARγ ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARγ ligand ciglitazone with an endogenous PPARγ ligand, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15‐deoxy PGJ2), 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 PPARγ mRNA is expressed in CGN cultures and observed ciglitazone‐ and 15‐deoxy PGJ2‐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 PGJ2 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 PGJ2. Despite the absence of neuroprotection, 15‐deoxy PGJ2 markedly inhibited the early up‐regulation of c‐Jun during KCl withdrawal. These studies suggest that ciglitazone and 15‐deoxy PGJ2 have markedly different effects on inherent and low‐KCl‐induced toxicity and c‐Jun expression in CGN, indicating potential non‐PPARγ mechanisms.