Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F.

Sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) is the most abundant store of intracellular Ca(2+), and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca(2+) in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca(2+) indicator, to directly monitor changes in RPTC ER Ca(2+). Fluo5F staining reflected ER Ca(2+), resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca(2+) pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca(2+) ionophore caused more rapid ER Ca(2+) release (55% and 75% decrease in fluorescence at 5 and 15 min). Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca(2+). In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca(2+) release. ER Ca(2+) release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca(2+) in live cells.

Functional analysis of the host defense peptide Human Beta Defensin-1: new insight into its potential role in cancer.

Although it is known that innate immunity is key for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have anti-tumor activity. Human Beta Defensin-1 (hBD-1), an important component of the innate immune response, is lost at high frequencies in malignant prostatic tissue, while high levels of expression are maintained in adjacent benign regions. In prostate carcinoma, frequent genetic alterations occur in the 8p22-23 region and several studies indicate there may be multiple tumor suppressor genes present within this region. The high incidence of loss of hBD-1 expression in prostate cancer, along with its chromosomal location of 8p23.2, raised the possibility that it may play a role in tumor suppression. To gain insight as to its function in prostate cancer, hBD-1 was cloned and ectopically expressed in four prostate cancer cell lines. Induction of hBD-1 expression resulted in a decrease in cellular growth in DU145 and PC3 cells. However, hBD-1 has no effect on the growth of androgen receptor (AR) positive LNCaP prostate cancer cells, but was again growth suppressive to PC3 cells with ectopic AR expression (PC3/AR+). hBD-1 also caused rapid induction of cytolysis and caspase-mediated apoptosis in DU145 and PC3 prostate cancer cells. Although the regulation of hBD-1 was not addressed in this study, our preliminary data demonstrated that the pathways involved may include cMYC and PAX2. Data presented here are the first to provide evidence of its potential role in prostate cancer cell death.

Inhibition of PAX2 expression results in alternate cell death pathways in prostate cancer cells differing in p53 status.

Inhibition of apoptosis is a critical pathophysiological factor that contributes to the development of prostate cancer. Recently, PAX2, a transcriptional regulator implicated in oncogenesis, has been demonstrated to be expressed by prostate cancer. However, its downstream molecular pathways for suppression of apoptosis, other than the tumor suppressor gene p53, have yet to be elucidated. Here, we examine the effects of inhibiting PAX2 expression by prostate cancer cells that differ in p53 gene status. These data collectively demonstrate that PAX2 inhibition results in cell death independent of p53, and that additional tumor suppressors or cell death pathways may be inhibited by PAX2 in prostate cancer cells.

The role of endoplasmic reticulum Ca2+-independent phospholipase a2γ in oxidant-induced lipid peroxidation, Ca2+ release, and renal cell death.

Oxidant-induced lipid peroxidation and cell death are major components of ischemia/reperfusion and toxicant injury. Our previous studies showed that renal proximal tubular cells (RPTCs) express Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)γ is cytoprotective. Our present studies reveal the role of ER-iPLA(2)γ in oxidant-induced ER lipid peroxidation, Ca(2+) release, and cell death. Oxidant tert-butyl hydroperoxide (TBHP) caused ER lipid peroxidation and Ca(2+) release in isolated rabbit kidney cortex microsomes. ER-iPLA(2)γ inhibition, using bromoenol lactone (BEL), potentiated both oxidant-induced ER lipid peroxidation and Ca(2+) release. Assessment of fatty acids using electrospray ionization-mass spectrometry revealed that ER-iPLA(2)γ mediates the TBHP-induced release of arachidonic acid (20:4), linoleic acid (18:2), and their oxidized forms (18:2-OH, 18:2-OOH, 20:4-OH, 20:4-OOH, 20:4-(OH)(3). iPLA(2)γ inhibition also accelerated oxidant-induced ER Ca(2+) release in RPTC. Depletion of ER Ca(2+) stores in RPTC with thapsigargin, an ER Ca(2+) pump inhibitor, prior to TBHP exposure reduced necrotic cell death and blocked the potentiation of TBHP-induced necrotic cell death by BEL. Together, these data provide strong evidence that ER-iPLA(2)γ protects renal cells from oxidant-induced necrotic cell death by releasing unsaturated and/or oxidized fatty acids from ER membranes, thereby preserving ER membrane integrity and preventing ER Ca(2+) release.