R. Chase Southard

Influence of serotonin on the kinetics of vesicular release

The mechanisms by which synaptic vesicles are transported and primed to fuse with the presynaptic membrane are important to all chemical synapses. Processes of signal transduction that affect vesicular dynamics, such as the second-messenger cascades induced by neuromodulators, are more readily addressed in assessable synaptic preparations of neuromuscular junctions in the crayfish. We assessed the effects of serotonin (5-HT) through the analysis of the latency jitter and the quantal parameters: n and p in the opener muscle of the walking leg in crayfish. There is an increase in the size of the postsynaptic currents due to more vesicles being released. Quantal analysis reveals a presynaptic mechanism by an increase in the number of vesicles being released. Latency measures show more events occur with a short latency in the presence of 5-HT. No effect on the frequency or size of spontaneous release was detected. Thus, the influence of 5-HT is presynaptic, leading to a release of more vesicles at a faster rate.

Down-regulation of PPARgamma1 suppresses cell growth and induces apoptosis in MCF-7 breast cancer cells

BackgroundPeroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARγ has been identified as a potential target for breast cancer therapy based on the fact that its activation by synthetic ligands affects the differentiation, proliferation, and apoptosis of cancer cells. However, the controversial nature of current studies and disappointing results from clinical trials raise questions about the contribution of PPARγ signaling in breast cancer development in the absence of stimulation by exogenous ligands. Recent reports from both in vitro and in vivo studies are inconsistent and suggest that endogenous activation of PPARγ plays a much more complex role in initiation and progression of cancer than previously thought.ResultsWe have previously demonstrated that an increase in expression of PPARγ1 in MCF-7 breast cancer cells is driven by a tumor-specific promoter. Myc-associated zinc finger protein (MAZ) was identified as a transcriptional mediator of PPARγ1 expression in these cells. In this study, using RNA interference (RNAi) to inhibit PPARγ1 expression directly or via down-regulation of MAZ, we report for the first time that a decrease in PPARγ1 expression results in reduced cellular proliferation in MCF-7 breast cancer cells. Furthermore, we demonstrate that these changes in proliferation are associated with a significant decrease in cell transition from G1 to the S phase. Using a dominant-negative mutant of PPARγ1, Δ462, we confirmed that PPARγ1 acts as a pro-survival factor and showed that this phenomenon is not limited to MCF-7 cells. Finally, we demonstrate that down-regulation of PPARγ1 expression leads to an induction of apoptosis in MCF-7 cells, confirmed by analyzing Bcl-2 expression and PARP-1 cleavage.ConclusionThus, these findings suggest that an increase in PPARγ1 signaling observed in breast cancer contributes to an imbalance between proliferation and apoptosis, and may be an important hallmark of breast tumorigenesis. The results presented here also warrant further investigation regarding the use of PPARγ ligands in patients who are predisposed or already diagnosed with breast cancer.

Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARγ Independent Manner

Background Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs—Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)—on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity. Principal Findings Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G0/G1 stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent. Conclusions CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle.

The Increased Expression of Peroxisome Proliferator-Activated Receptor-γ1 in Human Breast Cancer Is Mediated by Selective Promoter Usage

Peroxisome proliferator-activated receptor-γ1 (PPARγ1) is transactivated by a wide range of ligands in normal human mammary epithelial and breast cancer cells. Although transactivation of PPARγ mediates the expression of genes that are markers of differentiation, its overexpression in cancers of the breast, thyroid, colon, and lung suggests its dysregulation may play a role in oncogenesis, cancer progression, or both. We report the overexpression of PPARγ is caused by the use of a tumor-specific promoter in breast cancer cells that is distinct from the promoter used in normal epithelia. Thus, the increase in PPARγ expression seen in breast cancer cells results from promoter recruitment, providing new insights into the expression and actions of PPARγ in breast cancer.

Activation of the PKC pathway stimulates ovarian cancer cell proliferation, migration, and expression of MMP7 and MMP10.

ABSTRACT Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as luteinizing hormone (LH). Gonadotropins and other stimuli are capable of activating two pathways, PKA and PKC, that are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of human chorionic gonadotropin (hCG), an LH mimic, and an activator of the PKC pathway, phorbol-12-myristate 13-acetate (PMA), on ovarian cancer cell-cycle kinetics and apoptosis in Ovcar3 cells. PMA treatment increased cells in the S phase of the cell cycle and initially increased apoptosis after 4 h before diminishing apoptosis after 8 h. Treatment of ovarian cancer cells with hCG had no effect on these parameters. The PKC pathway is known to differentially regulate matrix metalloproteinase (MMP) expression. Results showed that ovarian cancer cells treated with PMA increased MMP7 and MMP10 mRNA levels after 8 h of treatment, and expression remained high after 12 h before decreasing at 24 h. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of PMA activation of MMPs on ovarian cancer cell migration. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration.

The complexity of signaling in host–pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation

The inhibition of apoptosis by Toxoplasma gondii is governed by its modulation of several signaling cascades including the NFkappaappaB and JNK pathways. This is evident in the dysregulation of JNK activation following treatment with UV and TNFalpha, both apoptogenic stimuli. Infection-mediated interference with the JNK cascade was found to be highly reproducible in HeLa cells. In light of emerging evidence regarding cross talk between the JNK and NFkappaB cascades, we examined the impact of infection in wild type and RelA/p65-/- mouse embryonic fibroblasts (MEF). Remarkably, parasite infection failed to significantly impact both UV and TNFalpha-mediated JNK phosphorylation in both cell lines suggesting a cell type specific effect. Furthermore siRNA-mediated knockdown of RelA/p65 failed to impact the parasite mediated effects on stimulus dependent activation of JNK in HeLa cells. Finally, the infection mediated suppression of JNK phosphorylation in HeLa cells did not result in decreased JNK kinase activity. Rather, the reduced levels of phospho-JNK in infected cells correlated with increased phosphatase activity noted by the partial rescue of the phenotype following treatment with okadaic acid. Taken together the results indicate that manipulation of the JNK pathway does not involve NFkappaB and is furthermore not a central component of the parasite enforced block of apoptosis. It further highlights the complexity of these systems and the danger of extrapolating results both within and across pathogen-host cell systems based on limited studies.

Influence of Neuromodulators and Vesicle Docking Related Proteins on Quantal Release

We assessed the mechanisms and related kinetics of vesicular release during the enhancement and depression of synaptic efficacy by the exogenous application of neuromodulators as well as altering the levels of protein related to vesicle fusion within nerve terminals. In order for a vesicle to be released, it must first dock at the presynaptic membrane; then, in the presence of Ca2+, a fusion pore for evoked transmitter release opens. To determine if the presence of synaptically relevant proteins, such as α-SNAP, can alter the number of evoked quantal units and then-rate of occurrence, α-SNAP was injected into the nerve terminals and the kinetics of release were measured. The enhancement of α-SNAP within the nerve terminal increases the number of rapidly occurring quantal events. The neuromodulator serotonin (5-HT), known to stimulate an IP3 cascade, also causes an enhancement of evoked quantal events. The presence of 5-HT or ot-SNAP at the terminal results in a faster rate of release for multiple events without a detectable change in the initial rate of primary events. Interestingly, the molt-related hormone, ecdysone (20-HE), causes a decrease in the rate of multiple events and results in an increase in latency jitter of vesicular release, thus indicating that 20-HE may act as a neuromodulator that depresses release. Investigating proteins and modulators of synaptic function will hopefully allow one to understand some of the mechanisms underlying synaptic differentiation and selective actions of neuromodulators, individually and in combination.