David M. Terrian

Protein kinase C ɛ interacts with Bax and promotes survival of human prostate cancer cells

Prostatic glandular epithelial cells express protein kinase Cɛ (PKCɛ), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCɛ might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCɛ, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors. An investigation into the biological significance of the PKCɛ association with Bax provided the first evidence of an inverse relationship between endogenous levels of PKCɛ and susceptibility of prostate cancer cells to the apoptotic effects of phorbol esters. Western blot and antisense experiments demonstrated that CWR-R1 cells expressed moderate levels of PKCɛ and relied on this protein to survive in the presence of phorbol esters, while the apoptosis normally induced by phorbol esters in PKCɛ-deficient LNCaP cells was dependent on the presence of Bax. Forced expression of PKCɛ in LNCaP cells was sufficient to confer a significant resistance to phorbol esters and this resistance was associated with an inhibition of phorbol ester-induced Bax conformational rearrangements that are important for Bax oligomerization, mitochondrial integration, and cytochrome c release. Considered in their entirety, our data suggest that an association of PKCɛ with Bax may neutralize apoptotic signals propagated through a mitochondrial death-signaling pathway.

Molecular Analysis of the Interactions between Protein Kinase C-ε and Filamentous Actin

Protein kinase C-epsilon (PKC-ε) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-ε and actin. Full-length recombinant PKC-ε, but not PKC-βII, -δ, -η, or -ζ, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-ε amino acids 222–230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-ε or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-ε colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-ε phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-ε sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-ε in a catalytically active conformation.

Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors.

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 over-expression

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15‐hydroperoxyeicosatetraenoic acid (15‐HPETE) or 13‐hydroperoxydodecadienoic acid (13‐HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15‐HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl‐2 proto‐oncogene were 6‐ to 8‐fold more resistant to apoptotic killing by tumor necrosis factor‐α, but only 0.4‐fold more resistant to 15‐HPETE. Thus, Bcl‐2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Abstract: Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin‐derived peptides and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+‐dependent manner by K+‐in‐duced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2‐chloro‐adenosine, inhibited the K+‐stimulated release of both dynorphin B and dynorphin A(l‐8). 2‐Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D‐aspartic acid to deplete the cytosolic, Ca2+‐independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+‐dependent pool of glutamic acid was significantly inhibited by NiCl2, 2‐chloroadenosine, 5′‐N‐ethylcarboxami‐doadenosine, cyclohexyladenosine, and R(‐)‐N6(2‐phenyl‐isopropyl)adenosine, but not by ATP. 2‐Chloroadenosine‐induced inhibition was reversed when the external CaCl2concentration was raised from 1.8 mMto 6 mM. 8‐Phen‐yltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2‐chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+‐evoked release of both glutamic acid and dynorphin A(l‐8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin‐derived peptides

Targeting prostate cancer based on signal transduction and cell cycle pathways

Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness.

U-50,488H INHIBITS DYNORPHIN AND GLUTAMATE RELEASE FROM GUINEA PIG HIPPOCAMPAL MOSSY FIBER TERMINALS

The selective kappa opioid agonist U-50,488H was tested for its ability to modulate the potassium-induced rise of cytosolic Ca2+ in, and transmitter release from, guinea pig hippocampal mossy fiber synaptosomes. U-50,488H dose dependently inhibited the potassium-induced rise in synaptosomal free Ca2+ levels. This inhibition was attenuated by the selective kappa opioid antagonist nor-binaltorphimine, but was insensitive to naloxone and the sigma opioid antagonist ICI 174,864. U-50,488H also dose dependently depressed the potassium-induced release of L-glutamate and dynorphin B-like immunoreactivity from mossy fiber synaptosomes in a nor-binaltorphimine-sensitive manner. This is the first report to confirm the presence of a presynaptic kappa opioid receptor in the hippocampal mossy fiber-CA3 synapse and the nature of its influence on neurotransmitter release. The present results may be used to suggest that endogenous dynorphin peptides interact with this kappa opioid receptor to autoregulate the excitatory mossy fiber synaptic input.

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.

A Dominant Role for p53-Dependent Cellular Senescence in Radiosensitization of Human Prostate Cancer Cells

Because p53 inactivation may limit the effectiveness of radiation therapy for localized prostate cancer, it is important to understand how this gene regulates clonogenic survival after an exposure to ionizing radiation. Here, we show that premature cellular senescence is the principal mode of cell death accounting for the radiosensitivity of human prostate cancer cell lines retaining p53 function. Alternative stress response pathways controlled by this tumor suppressor, including cell cycle arrest, DNA damage repair, mitotic catastrophe and apoptosis, contributed significantly less to radiation-induced clonogenic death. Using a dominant negative C-terminal fragment of p53, we present the first evidence that a complete loss of endogenous p53 function is sufficient to limit the irradiation-induced senescence and clonogenic death of prostate cancer cells. Conversely, inheritance of wild-type p53 by prostate cancer cells lacking a functional allele of this gene (i.e., DU145) significantly increases clonogenic death through p53-dependent cellular senescence and apoptotic pathways. Our data provide evidence that mutations of even one p53 allele may be sufficient to alter their clonogenic fate. In addition, they support the idea that the p53 pathway can be used as a specific target for enhancing the radiosensitivity of prostate cancer cells. Activation of p53 by the drug nutlin-3 is shown to be an effective radiosensitizer of prostate cancer cells retaining functional alleles of p53 and this effect was entirely attributable to an increased induction of p53-dependent cellular senescence.

Integrin signaling links protein kinase Cepsilon to the protein kinase B/Akt survival pathway in recurrent prostate cancer cells.

Failure of hormone therapy often involves an outgrowth of protein kinase Cɛ (PKCɛ)-positive cells in recurrent prostate cancer. Our previous investigations have uncovered evidence of a complex signaling network operating downstream of this oncogenic protein kinase to actively advance the survival and proliferation of prostate cancer cells. In this study, we present evidence of a functional interplay among integrin receptors, PKCɛ, and protein kinase B (PKB/Akt) in recurrent CWR-R1 prostate cancer cells. Flow cytometry and confocal microscopy provided evidence that PKCɛ signaling promoted the assembly of matrix adhesions containing an abundance of colocalized actin filaments and β1 integrins that exhibited an exposed activation epitope on the surface of live CWR-R1 cells. Reciprocal coimmunoprecipitations provided evidence of signaling complexes containing PKCɛ, β1 integrins, Src, and PKB/Akt in CWR-R1 cell cultures. An investigation into the functional significance of these interactions, and of their positive influence on β1 integrins, demonstrated that PKCɛ and several key components of the PKB/Akt signaling pathway remain constitutively phosphorylated/activated in adherent but not suspension cultures of PTEN-positive CWR-R1 cells. Gene transfer, antisense and pharmacological experiments provided additional support for the hypothesis that a mutually reinforcing signaling loop sustains the activation of β1 integrins, PKCɛ, and PKB/Akt in adherent prostate cancer cells.