Exposure to high-level noise leads to oxidative stress and triggers apoptosis of the hair cells. This study examined whether p53, a tumor suppressor protein, is activated in the cochlea following impulse noise exposure. Inhibition of p53 with pifithrin alpha, a specific p53 inhibitor, or KX1-004, a Src-protein tyrosine kinase inhibitor, was tested to determine if p53 inhibition could reduce noise-induced hearing loss and cochlear damage. Chinchillas were pre-treated with a local administration of pifithrin alpha or KX1-004 and exposed to impulse noise. The chinchillas were assessed for threshold shift at 1 and 24h after the noise. At 4 or 24h post noise, the cochleae were removed and organs of Corti were examined to assess the damage to the cells and upregulation of p53 by the noise. Apoptosis was evident in both outer hair cells and supporting cells. Phospho-p53 (Ser 15) was upregulated 4h and 24h after the noise. KX1-004 and pifithrin alpha both decreased threshold shift and the number of missing outer hair cells. These results indicate that p53 is involved in the early stages of noise-induced cell death and inhibition of this signaling pathway is a potential protective strategy against noise-induced hearing loss.

BACKGROUND:Serum prostate specific antigen (PSA) levels in prostate cancer patients serve as a useful biomarker for diagnosing and monitoring prostate cancer. Recently, secreted PSA has been characterized as an autocrine survival factor through activation of Akt and induction of AR. In the normal prostate, PSA is secreted in the lumen of prostatic ducts to lyse proteins in the seminal coagulum. METHODS:However, the mechanism for constitutive PSA secretion from benign prostate and its transport across the prostate-blood barrier into serum are unknown. Regulation of peptide secretion by iPLA(2)-beta has been reported in non-prostatic tissue and in prostate tissue iPLA(2)-beta is reported to be under androgen regulation. We investigated whether iPLA(2) plays a role for in PSA secretion by comparing iPLA(2) activity and expression in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. Expression of the two active iPLA(2)-beta mRNA splice variants, LH-iPLA(2) and SH-iPLA(2), were increased and the inhibitory ankyrin-iPLA(2) isoform was markedly reduced in LNCaP cells as compared to normal prostate epithelial RWPE-1 cells. RESULTS:These changes are consistent with a higher enzymatic activity in LNCaP cells. The iPLA(2)-beta-specific inhibitor BEL inhibited PSA secretion and induced apoptosis in LNCaP cells. iPLA(2) knockdown using SiRNA inhibited PSA secretion, downregulated AR and induced apoptosis. Exogenous PSA suppressed BEL-induced apoptosis and neutralizing anti-PSA antibody blocked the survival effect of PSA. CONCLUSIONS:These data indicate that iPLA(2)-beta participates in regulating PSA secretion and supports the concept that secreted PSA provides an autocrine survival function in LNCaP cells.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4401 PSA is an important biomarker for diagnosis of and prognosis assessment in prostate cancer. We have previously demonstrated that calcium-independent PLA2 (iPLA2) controls the secretion of PSA and inhibition of iPLA2 causes apoptosis in LNCaP cells. Addition of the PLA2 product arachidonic acid or its eicosanoid derivatives had no discernable effect on iPLA2-mediated apoptosis in LNCaP cells. However, addition of PSA together with iPLA2 inhibitor restored survival and addition of neutralizing antibody to PSA resulted in apoptosis. Addition of neutralizing antibody alone to LNCaP cells is sufficient to cause activation of caspases, indicative of apoptosis. Based on these data, we hypothesized that PSA plays an autocrine role that affects the survival of prostate cancer cells. Since the PI3K/Akt pathway plays a major role in prostate cancer cell survival, we tested whether PSA could activate the PI3K/Akt. Investigation of PSA signaling by western analysis shows that PSA causes the activation of Akt through phosphorylation at both Ser-473 and Thr-308 in LNCaP cells in a dose and time-dependent fashion. Phosphorylation of Akt was initiated within 2 hr by PSA at concentrations between 200 and 700 ng/ml and incubation at 3 hr required 25 to 200 ng/ml PSA. Treatment with PI3K inhibitors LY294002 and wortmannin in combination with PSA completed inhibited Akt activation. These data indicate that PSA participates in autocrine signaling that contributes to the regulation of survival of prostate cancer cells. While PSA is one of the best biomarkers for the early detection of any cancer, its effect on patient survival is under question. A role for PSA in cancer progression could explain its minimal impact on patient survival while acting as an early warning signal for the disease.

Thioflavin T (ThT) fluorescence is a commonly used method to monitor Abeta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1min and ThT does not interfere with aggregation of Abeta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of Abeta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between Abeta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower Abeta concentrations than is typically observed. In addition, differences in Abeta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against Abeta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify Abeta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize Abeta fibrils in a dose and time-dependent fashion.

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793 Serum prostate specific antigen (PSA) levels are elevated in prostate cancer patients and serve as a diagnostic marker for the disease. Prostate cancer is generally responsive to androgen ablation therapy and produces a drop in PSA levels. In time, hormone-refractory disease often develops and PSA levels once again rise. Under normal circumstances PSA is deposited in the lumen of prostatic ducts, whereas PSA secreted from malignant cells gains access to the circulation. The underlying biochemical mechanisms for the aberrant control of PSA secretion in prostate tumors are not well-known. Regulation of peptide secretion by iPLA2-β has recently been reported in non-prostatic tissue. In prostate tissue, both iPLA2-β and PSA expression are reportedly under androgen regulation. We therefore investigated whether iPLA2-β plays a role in PSA secretion in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. We observed an increase in iPLA2 activity in LNCaP cells that corresponded with an increase in expression of the two active isoforms, LH-iPLA2 and SH-iPLA2, as well as a decrease in the inhibitory ankyrin-iPLA2 isoform. We further demonstrated that treatment with the iPLA2-specfic inhibitor, BEL, inhibited PSA secretion from LNCaP cells and subsequently resulted in cell death. While BEL treatment triggered an apoptotic response in LNCaP cells, normal prostate RWPE-1epithelial cells were not vulnerable to BEL-induced cell death. Addition of exogenous PSA to LNCaP cultures suppressed BEL-induced cell death and addition of anti-PSA antibody reversed the survival effect of PSA. These data demonstrate that iPLA2 plays a role in regulating PSA secretion from cells and the release of PSA provides an autocrine survival function for LNCaP cells. The mechanism by which PSA imparts its survival effect is not currently understood.

We have reported the presence of OHC apoptosis and necrosis in the organ of Corti following exposure to intense noise. The current study was designed to investigate the rapidity and the initial pattern of outer hair cell (OHC) death induced by exposure to impulse noise. Chinchillas were exposed to 75 pairs of impulse noise at 155 dB peak sound pressure level presented over a time period of 75 s. At 5 or 30 min after the noise exposure, the cochleae were examined for morphological and biological indicators of apoptosis and necrosis. In the cochleae collected within 5 min after the 75-s noise exposure, there were clear signs of nuclear condensation and cell body shrinkage, suggesting the presence of OHC apoptosis. Apoptotic OHCs were further detected by positive staining of TUNEL and caspase-3 assays. In contrast to the rapid development of nuclear condensation, appearance of nuclear swelling, a necrotic phenotype, appeared at 30 min after the noise exposure. The results of the study demonstrate that induction of OHC apoptosis after the noise exposure is an extremely rapid process.

We have reported that by 2 days after noise exposure the size of cochlear lesion was expanding by outer hair cells (OHCs) dying either by apoptosis or necrosis [Hear. Res. 166 (2002) 62]. The current study was designed to compare the prevalence of the two cell death pathways as a function of time after exposure to noises of different levels. Chinchillas were exposed to a narrow band noise at either 104 or 108 dB SPL for 1 h. At three time points (1, 4 and 30 days) after the noise exposure, the numbers of missing, apoptotic and necrotic OHCs in the cochleas were identified and documented with a combination of TUNEL, caspase-3 and propidium iodide labeling. The subjects exposed to the 108-dB noise showed significantly more apoptotic OHCs than necrotic OHCs in the cochleas examined at days 1 and 4 after the noise exposure. By day 30, apoptotic and necrotic pathologies continued, although in small quantity, with no significant difference in quantity between two types of cell death. The subjects exposed to the 104-dB noise showed a significant difference in the numbers of apoptotic and necrotic OHCs at day 1 after the noise exposure, whereas the difference became statistically insignificant at day 4 and day 30 after the noise exposure. The results of the study indicate that the early expansion of cochlear lesion is attributed primarily to apoptosis, whereas the later stage of lesion expansion is likely the result of an equal contribution from apoptosis and necrosis.

derived neurotrophic factor 135 Brain plasticity 160

Some forms of ototoxicity appear to be mediated primarily by the superoxide radical; however, the exact role the superoxide radical plays in cochlear damage is not well understood because most ototoxic drugs produce multiple reactive oxygen species. To characterize the role of the superoxide radical in cochlear damage and the protective effect of compounds that inactivate superoxide, we treated mouse cochlear organotypic cultures for 24 h with paraquat, an herbicide that produces high levels of superoxide. M40403, a highly specific, nonpeptidyl mimetic of superoxide dismutase, was added to some cultures to inactivate the superoxide radical generated by paraquat. The number of outer hair cells (OHC) and inner hair cells (IHC) systematically decreased with increasing concentration of paraquat (0.01–10 mM). M40403 (10 µM) significantly increased OHC and IHC survival in cultures treated with 0.01–1.0 mM of paraquat. These results suggest that excess production of superoxide radical is a sufficient condition for hair cell loss.

We previously reported that intense noise exposure causes outer hair cell (OHC) death primarily through apoptosis. Here we investigated the intracellular signal pathways associated with apoptotic OHC death. Chinchillas were exposed to a 4 kHz narrowband noise at 110 dB SPL for 1 h. After the noise exposure, the cochleas were examined for the activity of each of three caspases, including caspase-3, -8, or -9 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleas were further examined for cytochrome c release from mitochondria by immunohistology and for DNA degradation by the TUNEL method. The results showed that the noise exposure triggered activation of caspase-3, an important mediator of apoptosis. The noise exposure also caused the activation of caspase-8 and caspase-9, each of which is associated with a distinct signaling pathway that leads to activation of caspase-3. Caspase activation occurred only in the apoptotic OHCs and not in the necrotic OHCs. These results indicate that multiple signaling pathways leading to caspase-3 activation take place simultaneously in the apoptotic OHCs. In addition to caspase activation, noise exposure caused the release of cytochrome c from mitochondria, resulting in a punctate fluorescence in the cytosol. In contrast to activation of caspases, the release of cytochrome c took place in both apoptotic and necrotic OHCs. Moreover, the release of cytochrome c in a subpopulation of OHCs took place early in the cell death process, prior to any outward signs of necrosis or apoptosis. These data suggest that in this subpopulation there exists a common step that is shared by cell death pathways before entering either necrosis or apoptosis. Lastly, use of the TUNEL assay in combination with PI labeling provides a more accurate discrimination between apoptosis and necrosis.

Permanent hearing loss by noise exposure is the functional consequence of a complex set of pathological changes in the cochlea. Among these changes, hair cell (HC) death may contribute most to the loss of auditory function. It has been known that noise-induced HC death starts during a noise exposure and continues even after the termination of the noise exposure. Biological mechanisms underlying the progression of HC death are not fully understood. Here we describe progression of HC death in the chinchilla cochlea after exposure to a 4-kHz narrow band noise at 110 dB sound pressure level (SPL) for 1 hour. Morphological examination of HC nuclei revealed typical nuclear changes for both apoptosis and necrosis. Apoptosis appeared to be a major death pathway leading to progression of the cochlear lesion in noise-damaged cochleas. The study also showed that expansion of HC death developed asymmetrically toward the apical and basal parts of the cochleas. Detection of caspase-3 activation showed a spatial agreement between the apoptotic nuclear changes and caspase-3 activation. These results clearly implicate the apoptotic pathway in the postexposure progression of HC demise.

It has been known for some time that noise-induced outer hair cell (OHC) death in the cochlea continues well after the termination of a noise exposure. However, the underlying mechanisms leading to the expansion of a cochlear lesion are not fully understood. Here we report involvement of the apoptotic pathway in the progression of OHC death in the chinchilla cochlea following exposure to a 4 kHz narrow band noise at 110 dB SPL for 1 h. Morphological examination of OHC nuclei revealed nuclear condensation and fragmentation, typical morphological features of apoptosis. OHC apoptosis developed asymmetrically toward the apical and basal parts of the cochleas following the noise exposure. Two days after the noise exposure, there was still active OHC pathology with condensed and fragmented nuclei in the basal part of the cochleas. Detection of caspase-3 activation, an intracellular marker for apoptosis, showed a spatial agreement between the apoptotic nuclei and activated caspase-3. These results clearly implicate the apoptotic pathway in the post-exposure progression of OHC demise.

It has been known for some time that noise-induced outer hair cell (OHC) death in the cochlea continues well after the termination of a noise exposure. However, the underlying mechanisms leading to the expansion of a cochlear lesion are not fully understood. Here we report involvement of the apoptotic pathway in the progression of OHC death in the chinchilla cochlea following exposure to a 4 kHz narrow band noise at 110 dB SPL for 1 h. Morphological examination of OHC nuclei revealed nuclear condensation and fragmentation, typical morphological features of apoptosis. OHC apoptosis developed asymmetrically toward the apical and basal parts of the cochleas following the noise exposure. Two days after the noise exposure, there was still active OHC pathology with condensed and fragmented nuclei in the basal part of the cochleas. Detection of caspase-3 activation, an intracellular marker for apoptosis, showed a spatial agreement between the apoptotic nuclei and activated caspase-3. These results clearly implicate the apoptotic pathway in the post-exposure progression of OHC demise.

Apoptosis is an active cell death pathway involved in a variety of pathological conditions, including noise-induced outer hair cell (OHC) death. During this process, the cytoskeletal proteins have been found to be either damaged and/or enzymatically disassembled in several cell types, leading to formation of apoptotic manifestations. This study was designed to examine the cleavage of filamentous actin (F-actin), an important cytoskeletal protein, in the cochlear OHCs after noise exposure. Chinchillas were exposed to a 4 kHz narrow band noise at 106 dB SPL for 1 h and cochleas were either collected immediately or 3 h after the noise exposure. The organs of Corti were double-stained using FITC-labeled phalloidin for F-actin and propidium iodide for OHC nuclei. The effect of noise on F-actin and nuclei was examined by confocal microscopy. The result showed that the fluorescence associated with F-actin was decreased in the OHCs possessing condensed nuclei, but remained unchanged in the OHCs with swollen nuclei. The change in F-actin labeling occurred coordinately with the changes in nuclear morphology of apoptotic cells and was prevented by administration of caspase-3 inhibitor (Z-DEVD-FMK). The results of this study indicate that F-actin cleavage is an important early cellular event in apoptotic development in OHCs following exposure to traumatic noise.

We studied the reaction between hypochlorite anion (−OCl) and hydrogen peroxide (H2O2) by using spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The obtained data demonstrate that hydroxyl radical is produced in this reaction. Thus, −OCl and H2O2 could assume new relevance in environmental chemistry by representing a potential source of hydroxyl radicals and other oxidant species, such as HO–OCl and OCl radical.

Oxidative stress has been implicated in the etiology of many pathological states and known to result in DNA damage. Oxidative DNA damage can lead to mutagenesis (, , ) and has been associated with aging (), diabetes mellitus (), inflammatory disease () and carcinogenesis (, , ,). The 8-hydroxy-2-deoxyguanosine lesion (8-OHdG) is often used in the assessment of oxidative DNA damage and has become the de facto marker for oxidative damage to DNA. 8-OHdG is one of the most prominent lesions observed following exposure to ionizing radiation () but also results from treatment with many xenobiotics () as well as by endogenous mechanisms. Thus, normal endogenous levels becomes a critical issue in the assessment of cellular 8-OHdG levels in pathological states. Furthermore, 8-OHdG is efficiently repaired by a DNA glycosylase specific for this lesion () and its contribution to mutagenesis is relatively weak (, , ). However, it is representative of approximately 20 additional oxidatively-denved lesions known to result from radiation damage () and whose mutagenic outcome are largely unknown in mammalian-cell systems.