Biomedical subjects

Stephen J Beebe

Publications and source records attributed to Stephen J Beebe.

4 recordsLinked to original sources

PubMed2003

Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells.

Electroporation by using pulsed electric fields with long durations compared with the charging time of the plasma membrane can induce cell fusion or introduce xenomolecules into cells. Nanosecond pulse power technology generates pulses with high-intensity electric fields, but with such short durations that the charging time of the plasma membrane is not reached, but intracellular membranes are affected. To determine more specifically their effects on cell structure and function, human cells were exposed to high intensity (up to 300 kV/cm) nanosecond (10-300 ns) pulsed electric fields (nsPEF) and were analyzed at the cellular and molecular levels. As the pulse duration decreased, plasma membrane electroporation decreased and appearances of apoptosis markers were delayed. NsPEF induced apoptosis within tens of minutes, depending on the pulse duration. Annexin-V binding, caspase activation, decreased forward light scatter, and cytochrome c release into the cytoplasm were coincident. Apoptosis was caspase- and mitochondria-dependent but independent of plasma membrane electroporation and thermal changes. The results suggest that with decreasing pulse durations, nsPEF modulate cell signaling from the plasma membrane to intracellular structures and functions. NsPEF technology provides a unique, high-power, energy-independent tool to recruit plasma membrane and/or intracellular signaling mechanisms that can delete aberrant cells by apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2003

The effects of intense submicrosecond electrical pulses on cells.

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

Cell Membrane↗

PubMed2002

Resveratrol induces apoptosis in LNCaP cells and requires hydroxyl groups to decrease viability in LNCaP and DU 145 cells.

BACKGROUND: This study was conducted to determine the effects of resveratrol on prostate cancer cell viability through apoptosis induction and the significance of the three hydroxyl groups on resveratrol to the measured effect. METHODS: Hormone-sensitive LNCaP cells and hormone-insensitive DU 145 cells were treated with resveratrol, tri-methoxy-resveratrol, or diethylstilbestrol (DES; the positive control for toxicity and apoptosis). Cell viability was determined by using an MTS assay. Apoptosis was determined by the appearance of apoptotic morphology, annexin V-FITC-positive intact cells, and caspase activation. RESULTS: Resveratrol and DES decreased viability in LNCaP cells, but only resveratrol-treated cells expressed apoptotic morphology, annexin V-FITC-positive cells, and caspase activation. Tri-methoxy-resveratrol had no effect on DU 145 cell-viability and was less toxic to LNCaP cells than resveratrol. CONCLUSION: Resveratrol was toxic to cells regardless of whether the cells were hormone-responsive or -unresponsive. This finding suggests that the cell's hormone responsive status is not an important determinant of the response to resveratrol. Furthermore, the hydroxyl-groups on resveratrol are required for cell toxicity. Finally resveratrol but not DES induced caspase-mediated apoptosis.

Antineoplastic Agents, Hormonal↗

PubMed2002

Differential transcriptional regulation by the alpha- and gamma-catalytic subunit isoforms of cAMP-dependent protein kinase.

The C gamma and C alpha isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to C alpha, C gamma has a different substrate specificity and a selective pseudosubstrate specificity, exhibiting inhibition by regulatory subunits, but not by the protein kinase inhibitor. In these studies, C gamma-mediated gene transcription regulation was compared with that of C alpha in four cell lines using transient transfection/dual luciferase assays. As compared to C gamma, C alpha more efficiently activated a cAMP-response element (CRE)-regulated fragment of the human alpha-glycoprotein hormone promoter which was coupled to a firefly luciferase reporter gene (pGH alpha-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, C gamma, but not C alpha, activated the Sp1RE-regulated herpes simplex virus thymidine kinase promoter which was coupled to a Renilla luciferase reporter (pTK-rluc). In Sp1-deficient Sf9 cells, pGH alpha-fluc expression was maintained for both isoforms, but cotransfection with an Sp1 expression plasmid was necessary and sufficient for activation of pTK-rluc expression by C gamma. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both C alpha and C gamma (1.5- to 3-fold), while a catalytically inactive PDK1 mutant (PDK.KD) did not. These results suggest that both C alpha and C gamma can activate CRE-responsive genes; however, C alpha does so with better efficiency than C gamma. In contrast to C alpha, C gamma activates transcription of genes containing pTK-like Sp1RE sites. Activation of different C subunit isoforms can provide a means to diversify cAMP-mediated transcription, possibly affecting cell phenotype.

3-Phosphoinositide-Dependent Protein Kinases↗