Alterations in muscarinic receptor-coupled phosphoinositide hydrolysis and AP-1 activation in Alzheimer's disease cybrid cells.

Alzheimer's disease cybrid cells produced by replacing endogenous mitochondria in human neuroblastoma SH-SY5Y cells with platelet mitochondria from subjects with Alzheimer's disease have higher levels of reactive oxygen species than do cybrid cells with mitochondria from control subjects. These cells were used to test if this chronic mild increase in reactive oxygen species affects muscarinic receptor-coupled signaling activities. Basal and carbachol-stimulated phosphoinositide hydrolysis were higher, and there was less inhibition by glutathione depletion, in Alzheimer's disease than control cybrid cells. Elevated phosphoinositide hydrolysis in Alzheimer's disease cybrid cells also was evident upon direct activation of G-proteins (Gq/11) linked to phosphoinositide signaling or of phospholipase C, but immunoblot analyses revealed equivalent levels of Gq/11 and phospholipase C in both cell lines. These results indicate that there is up-regulation of phosphoinositide signaling in Alzheimer's disease cybrid cells in association with chronic mild oxidative stress, although treatment of cells with H(2)O(2) to induce greater acute oxidative stress caused decreases in carbachol-stimulated phosphoinositide hydrolysis that were similar in Alzheimer's disease and control cybrid cells. In contrast to phosphoinositide hydrolysis, carbachol-stimulated AP-1 DNA binding activity was lower in Alzheimer's disease than control cybrid cells, and this deficit was associated with deficient protein kinase C-mediated activation of AP-1. Overall, these results demonstrate that chronically elevated reactive oxygen species in Alzheimer's disease cybrid cells are associated with a more robust phosphoinositide signaling system, but lower signaling to activation of AP-1. These alterations may represent adaptations to exposure to oxidants, which precede more widespread deficits in signaling associated with more severe oxidative stress.

Rapid activation of heat shock factor-1 DNA binding by H2O2 and modulation by glutathione in human neuroblastoma and Alzheimer's disease cybrid cells.

Because cellular signaling systems are critical mediators of responses to oxidative stress, a condition associated with neurodegenerative disorders, the redox-dependent regulation of heat shock factor-1 (HSF-1) was investigated in human neuroblastoma SH-SY5Y cells. Exposure of cells to 200 microM H2O2 caused a rapid increase in HSF-1 DNA binding that was evident within 10 min, and caused a robust increase that reached levels 8-fold the basal activity. In comparison, the transcription factors, activator protein-1 (AP-1) and early growth response-1 (EGR-1), were activated more slowly and to a lesser extent. Activation of HSF-1 DNA binding activity was associated with a cytosolic to nuclear translocation of HSF-1 protein, and was detected with concentrations of H2O2 of 100 microM and greater. Intracellular glutathione modulated H2O2-induced HSF-1 DNA binding activity, as depletion of glutathione caused HSF-1 to be activated with lower concentrations of H2O2 (25 microM) and supplementation of glutathione blocked HSF-1 activation by 100 to 400 microM H2O2. Alzheimer's disease (AD) and control cybrid cells (SH-SY5Y cells in which the mitochondria were replaced with platelet mitochondria from AD or matched control subjects) were used to test the effects of the chronic oxidative stress caused by the excessive production of reactive oxygen intermediates (ROIs) in AD cybrids on HSF-1 activity. Basal and maximal (induced by H2O2 in glutathione-depleted cells) HSF-1 DNA binding activity were lower in AD than control cybrids, suggesting that the cells had compensated for excessive ROIs. These results indicate that the activation of HSF-1 is highly sensitive to oxidative stress and is regulated by endogenous antioxidant mechanisms.

Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium.

The compound 1-methyl-4-phenylpyridinium (MPP) is a selective inhibitor of mitochondrial complex I, and is widely used in model systems to elicit neurochemical alterations that may be associated with Parkinson's disease. In the present study treatment of human neuroblastoma SH-SY5Y cells with MPP resulted in a time- and concentration-dependent activation of the apoptosis-associated cysteine protease caspase-3, and caused morphological changes characteristic of apoptosis. To test if the activation state of the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway affects MPP-induced caspase-3 activation, PI3K was inhibited with LY294002, or activated with insulin-like growth factor-1. MPP-induced caspase-3 activation was increased by inhibition of PI3K, and decreased by stimulation of PI3K, indicative of anti-apoptotic signaling by the PI3K/Akt pathway. To test if glycogen synthase kinase-3beta (GSK3beta), a pro-apoptotic kinase that is inhibited by Akt, is involved in regulating MPP-induced apoptosis, overexpression of GSK3beta and lithium, a selective inhibitor of GSK3beta, were used to directly alter GSK3beta activity. MPP-induced caspase-3 activity was increased by overexpression of GSK3beta. Conversely, the GSK3beta inhibitor lithium attenuated MPP-induced caspase-3 activation. To test if these regulatory interactions applied to other mitochondrial complex I inhibitors, cells were treated with rotenone. Rotenone-induced activation of caspase-3 was enhanced by inhibition of PI3K or increased GSK3beta activity, and was attenuated by inhibiting GSK3beta with lithium. Overall, these results indicate that inhibition of GSK3beta provides protection against the toxic effects of agents, such as MPP and rotenone, that impair mitochondrial function.

Cholinergic- and stress-induced signaling activities in cells overexpressing wild-type and mutant presenilin-1.

This study examined the effects of overexpression of presenilin-1 wild-type (PS1wt) or mutant L286V (PS1m) in human neuroblastoma SH-SY5Y cells on signal transduction systems. Oxotremorine-M-induced activation of AP-1 was 40--53% lower in PS1wt than control cells, and further impaired (63--76%) in PS1m cells. Heat shock (45 degrees C) activated Akt, increased heat shock factor-1 (HSF-1) DNA binding activity, and increased levels of heat shock protein 70, and these responses were not altered by overexpression of PS1wt or PS1m. H(2)O(2) also caused a time-dependent increase in HSF-1 DNA binding activity which was similar in all cell lines. Thus, overexpression of PS1wt reduced muscarinic receptor-mediated activation of AP-1, and PS1m overexpression caused greater inhibition, but stress-induced activation of Akt and HSF-1 was unaffected by either PS1wt or PS1m.

Lead and mercury mutagenesis: role of H2O2, superoxide dismutase, and xanthine oxidase.

It has been suggested that reactive oxygen intermediates (ROIs) may have a role in the genotoxic effects of lead (Pb2+) and mercury (Hg2+), but there have not been any definitive studies demonstrating a causal relationship between the induction of ROIs by these metals and mutagenesis. We previously demonstrated, using the transgenic Chinese hamster ovary cell line AS52, that low concentrations (0.1-1 microM) of Pb2+ and Hg2+ are mutagenic. In the present study, using a novel histochemical computer-enhanced image analysis technique, we demonstrate that Pb2+ and Hg2+ induce the formation of H2O2 in AS52 cells by at least two distinct mechanisms. One is characterized by the rapid induction of H2O2 following treatment of cells with concentrations of Pb2+ or Hg2+ below 0.8 and 1 microM, respectively, while the second occurs in AS52 cells treated with concentrations of Pb2+ or Hg2+ greater than 0.8 and 1 microM, respectively. Pb2+ and Hg2+ (0.1-1 microM) had no effect on the activities of partially purified catalase, glutathione peroxidase, or glutathione reductase, important enzymes involved with antioxidant defense, but these metals stimulated the activities of copper-zinc superoxide dismutase (CuZn-SOD) and xanthine oxidase (XO). Allopurinol (50 microM), a specific inhibitor of xanthine oxidase, inhibited the induction of H2O2 by Pb2+ (0.8-1 microM) and Hg2+ (1 microM) and also inhibited Pb2+- and Hg2+-induced mutagenesis. These results demonstrate that Pb2+ and Hg2+ disrupt the redox status of AS52 cells by enhancing the activities of CuZn-SOD and XO. Furthermore, the results of these studies also demonstrate that there is a causal relationship between the induction of H2O2 by these metals and mutagenesis.

Ascorbic acid-dehydroascorbate induces cell cycle arrest at G2/M DNA damage checkpoint during oxidative stress.

Reactive oxygen species induce cellular damage and have been implicated as mediators for cellular signaling pathways. However, a linkage between the cellular redox status and cell cycle progression has not been demonstrated. We previously demonstrated, using the Chinese hamster ovary cell line AS52, that the cytotoxic and mutagenic effects of oxidative stress is prevented by ascorbic acid (AA), but only when cells are treated with AA prior to treatment with the stressor. To elucidate the mechanism(s) responsible for this effect, we determined the effect of AA on cell cycle progression during oxidative stress. Flow cytometric analyses demonstrated that treatment of AS52 cells with AA (50 microM), prior to treatment with a radical generating system (RGS), enhanced cell cycle arrest at the G2/M DNA damage checkpoint when compared to cells treated with RGS. AA had no effect on cell cycle progression in the absence of oxidative stress. Furthermore, under conditions that prevent the reduction of dehydroascorbate (DHA), the oxidized form of AA, cell cycle arrest was also induced at the G2/M DNA damage checkpoint. These observations demonstrate that during periods of oxidative stress, AA functions as an antioxidant and DHA enhances transient arrest at the G2/M checkpoint by delaying the activation of cyclin B-cdc2. These results suggest the presence of a unique redox mechanism for the regulation of cell cycle progression and also demonstrate a novel mechanism by which AA protects cells from damage due to oxidative stress.

Antimutagenic and promutagenic activity of ascorbic acid during oxidative stress.

Ascorbic acid (AA) has both antioxidant and prooxidant activities. However, there have not been any studies to elucidate the molecular mechanisms that determine whether AA functions as an anti- or a prooxidant during oxidative stress. The results of this study, using the Chinese hamster ovary cell line AS52 as a model system, demonstrate that there is a temporal relationship between the anti- and prooxidant activities of a physiologically relevant concentration of AA (50 microM) and oxidative stress. Treatment of cells with AA (50 microM) 24 hr prior to treatment of the cells with a radical generating system (RGS) results in a statistically significant inhibition of the cytotoxicity and mutagenicity associated with exposure of AS52 cells to oxidative stress. Conversely, cotreatment of cells with AA and the RGS results in a statistically significant increase in both the cytotoxic and mutagenic effects of oxidative stress when compared to cell populations exposed only to the RGS. The results, using a novel histochemical-computer image analysis system to detect hydrogen peroxide (H2O2), also demonstrate that there is a direct correlation between the ability of AA to decrease the levels of H2O2 in cells and the cytotoxic and mutagenic effects of oxidative stress. This study suggests that the time at which AA is administered in relation to exposure to oxidative stress has an impact on AA antimutagenic activity, and this may explain the conflicting results concerning the effectiveness of AA as a cancer chemopreventive agent.

Characterization of a murine model of acute lung injury (ALI): a prominent role for interleukin-1.

This report describes a model developed to study local and systemic events that occur as a result of acute lung injury (ALI). C57BL/6J mice were injected with a single intravenous dose (2, 4, and 6 micrograms) of 12-O-tetradecanoylphorbol-13-acetate (TPA). At 1, 2, 4, 12, 24, and 48 h, after injection, plasma was collected by sinus orbital puncture, bronchoalveolar lavage (BAL) was performed and cells and fluid were collected, lungs were perfused, and pulmonary tissue was isolated and processed for histological, immunochemical, and gene expression studies. The results indicate a dose-dependent increase in animal distress and a decrease in survival. TPA induced an early systemic response, reflected as an initial decrease in numbers of peripheral blood neutrophils at 1 h, followed at 2 h by a sustained increase. There was dose- and time-dependent increase in IL-1 beta mRNA synthesis, detected using RT-PCR, and in immunoreactive IL-1 alpha produced by both tissue-fixed pulmonary cells and cells within alveolar spaces. Infiltration of neutrophils into pulmonary tissue and increased protein content in BAL fluid was detected 2 h after injection of TPA. Disruptions in pulmonary architecture accompanied by the presence of highly vacuolated macrophages within the alveolar spaces and interstitial tissue were evident after IV injection of TPA. The study shows that injection of TPA induces reproducible dose- and time-dependent alterations in cell types, numbers, state of activation, and production of soluble mediators in the peripheral circulation within BAL and pulmonary tissue. Thus, this model offers a means to examine the cellular basis for the local and systemic alterations observed during ALI.

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma.

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

Transforming growth factor-alpha overexpression in proliferative verrucous leukoplakia and oral squamous cell carcinoma: an immunohistochemical study.

Proliferative verrucous leukoplakia is a unique type of oral leukoplakia that has a high risk of malignant transformation. The aim of this study was to examine the expression of transforming growth factor-alpha in proliferative verrucous leukoplakia, oral squamous cell carcinoma, and normal mucosa. Transforming growth factor-alpha, a potent mitogen, is known to play an important role in various neoplasms including oral squamous cell carcinoma. Immunohistochemical localization of transforming growth factor-alpha in archival paraffin-embedded sections was performed with commercially available monoclonal antibodies. Ten cases each of normal mucosa, proliferative verrucous leukoplakia, and oral squamous cell carcinoma were stained. Quantification of the staining intensity, expressed as the cytoplasmic optical density, was done with the Roche Image Analysis System. The data were statistically analyzed with the one-way analysis of variance and Tukey tests. Notably, the mean cytoplasmic optical density of proliferative verrucous leukoplakia was significantly higher than the mean cytoplasmic optical density of normal mucosa (p < 0.01). The mean cytoplasmic optical density of proliferative verrucous leukoplakia was slightly higher than that of oral squamous cell carcinoma, however, this difference was not significant (p > 0.05). The mean cytoplasmic optical density values demonstrate that increased transforming growth factor-alpha immunoreactivity occurs in proliferative verrucous leukoplakia and oral squamous cell carcinoma relative to normal mucosa.

Opposing actions of phosphatidylinositol 3-kinase and glycogen synthase kinase-3beta in the regulation of HSF-1 activity.

Elevated temperatures activate the survival promoters Akt and heat shock factor-1 (HSF-1), a transcription factor that induces the expression of heat shock proteins (HSPs), such as HSP-70. Because neuronal mechanisms controlling these responses are not known, these were investigated in human neuroblastoma SH-SY5Y cells. Heat shock (45 degrees C) rapidly activated Akt, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but only Akt was activated in a phosphatidylinositol 3-kinase (PI-3K)-dependent manner, as the PI-3K inhibitors LY294002 and wortmannin blocked Akt activation, but not ERK1/2 or p38 activation. Akt activation was not blocked by inhibition of p38 or ERK1/2, indicating the independence of these signaling systems. Heat shock treatment also caused a rapid increase in HSF-1 DNA binding activity that was partially dependent on PI-3K activity, as both the PI-3K inhibitors attenuated this response. Because Akt inhibits glycogen synthase kinase-3beta (GSK-3beta), an enzyme that facilitates cell death, we tested if GSK-3beta is a negative regulator of HSF-1 activation. Overexpression of GSK-3beta impaired heat shock-induced activation of HSF-1, and also reduced HSP-70 production, which was partially restored by the GSK-3beta inhibitor lithium. Thus, heat shock-induced activation of PI-3K and the inhibitory effect of GSK-3beta on HSF-1 activation and HSP-70 expression imply that Akt-induced inhibition of GSK-3beta contributes to the activation of HSF-1.

Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta.

The goal of this study was to determine whether the intracellular distribution of the proapoptotic enzyme glycogen synthase kinase-3 beta (GSK-3 beta) is dynamically regulated by conditions that activate apoptotic signaling cascades. In untreated human neuroblastoma SH-SY5Y cells, GSK-3 beta was predominantly cytosolic, although a low level was also detected in the nucleus. The nuclear level of GSK-3 beta was rapidly increased after exposure of cells to serum-free media, heat shock, or staurosporine. Although each of these conditions caused changes in the serine 9 and/or tyrosine phosphorylation of GSK-3 beta, neither of these modifications was correlated with nuclear accumulation of GSK-3 beta. Heat shock and staurosporine treatments increased nuclear GSK-3 beta prior to activation of caspase-9 and caspase-3, and this nuclear accumulation of GSK-3 beta was unaltered by pretreatment with a general caspase inhibitor. The GSK-3 beta inhibitor lithium did not alter heat shock-induced nuclear accumulation of GSK-3 beta but increased the nuclear level of cyclin D1, indicating that cyclin D1 is a substrate of nuclear GSK-3 beta. Thus, the intracellular distribution of GSK-3 beta is dynamically regulated by signaling cascades, and apoptotic stimuli cause increased nuclear levels of GSK-3 beta, which facilitates interactions with nuclear substrates.

Mood stabilizers, glycogen synthase kinase-3beta and cell survival.

Glycogen synthase kinase-3beta (GSK3beta) is a central figure in many intracellular signaling systems and is directly regulated by lithium. Substantial evidence now indicates that an important property of the mood stabilizer, lithium, is to influence GSK3beta-linked signaling pathways. This raises the possibility that other mood stabilizers act in a similar manner, which may include modulation of signaling systems leading to GSK3beta, direct regulation of GSK3beta or regulation of signaling intermediates downstream of GSK3beta. Downstream targets of GSK3beta, and thus potential targets of mood stabilizers, are several key transcription factors, including beta-catenin, AP-1, cyclic AMP-response element binding protein, NFkappaB, Myc, heat shock factor-1, nuclear factor of activated T-cells and CCAAT/enhancer-binding proteins. GSK3beta also is an important modulator of cell death, which may be a consequence of its regulatory effects on transcription factor activities. GSK3beta facilitates apoptosis, and lithium's inhibition of GSK3beta supports cell survival. Thus, signaling systems determining cell fate appear to be important targets of mood stabilizers, and these may include signaling pathways encompassing GSK3beta, including transcription factors regulated by GSK3beta.

Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium.

The potential role of glycogen synthase kinase-3beta in modulating apoptosis was examined in human SH-SY5Y neuroblastoma cells. Staurosporine treatment caused time- and concentration-dependent increases in the activities of caspase-3 and caspase-9 but not caspase-1, increased proteolysis of poly(ADP-ribose) polymerase, and induced morphological changes consistent with apoptosis. Overexpression of glycogen synthase kinase-3beta to levels 3.5 times that in control cells did not alter basal indices of apoptosis but potentiated staurosporine-induced activation of caspase-3, caspase-9, proteolysis of poly(ADP-ribose) polymerase, and morphological changes indicative of apoptosis. Inhibition of glycogen synthase kinase-3beta by lithium attenuated the enhanced staurosporine-induced activation of caspase-3 in cells overexpressing glycogen synthase kinase-3beta. In cells subjected to heat shock, caspase-3 activity was more than three times greater in glycogen synthase kinase-3beta-transfected than control cells, and this potentiated response was inhibited by lithium treatment. Thus, glycogen synthase kinase-3beta facilitated apoptosis induced by two experimental paradigms. These findings indicate that glycogen synthase kinase-3beta may contribute to pro-apoptotic-signaling activity, that inhibition of glycogen synthase kinase-3beta can contribute to anti-apoptotic-signaling mechanisms, and that the neuroprotective actions of lithium may be due in part to its inhibitory modulation of glycogen synthase kinase-3beta.

Transient oxidative stress in SH-SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis.

The effects of an oxidative insult on cell survival and tau metabolism were investigated in human neuroblastoma SH-SY5Y cells. In this treatment paradigm cells were exposed to the membrane permeant oxidant tert-butylhydroperoxide (tBHP) for 40 min, returned to fresh media and cell survival/death was monitored during the post-treatment period. Cell viability decreased significantly by 6 hr after tBHP exposure, and by 24 hr lactate dehydrogenase (LDH) release was 40.1 +/- 8.8% in tBHP treated cells compared to 8.1 +/- 4.7% in control cells. This oxidative stress paradigm also resulted in significant activation of caspase-3 by 2 hr post-treatment and nuclear apoptotic morphology. Furthermore, tBHP treatment also resulted in delayed tau proteolysis that was first evident 2 hr post-treatment. Treatment of the cells with the general caspase inhibitor Boc-Asp(OMe)-Fluoromethylketone (BAF) completely inhibited caspase-3 activation in response to tBHP, and delayed, but did not prevent cell death. BAF treatment also decreased tau proteolysis. In vitro, recombinant tau was readily proteolyzed by active recombinant caspase-3 into a stable breakdown product. Further tau in the cell lysates was cleaved by active recombinant caspase-3 at a rate, and to an extent similar to that observed for the well-established caspase-3 substrate poly(ADP-ribose)polymerase (PARP). These results suggest that oxidative stress-induced cell death occurs through both caspase-dependent and-independent pathways, and that tau is likely an in situ substrate of caspase-3.

Interleukin-1 alpha gene expression and localization of interleukin-1 alpha protein during tumor promotion.

Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1 alpha (IL-1 alpha) gene expression. Levels of IL-1 alpha mRNA were elevated as early as 15 min and peaked at 3-4 h after a single application of TPA (2 micrograms or 10 micrograms). IL-1 alpha gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 micrograms TPA. IL-1 alpha-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1-22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1 alpha-immunoreactive protein. DMBA treatment alone did not induce IL-1 alpha gene expression. Injection of IL-1 alpha-specific antibodies (50 micrograms) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 micrograms or 10 micrograms) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti-IL-1 alpha antibodies inhibited incorporation of [methyl-3H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1 alpha is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1 alpha primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1 alpha may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.

Alterations in transforming growth factor-alpha and epidermal growth factor receptor expression during rat esophageal tumorigenesis.

Transforming growth factor-alpha (TGF-alpha) stimulates cell proliferation through interaction with its receptor, the epidermal growth factor receptor (EGFR), by activating its tyrosine kinase activities. The simultaneous overexpression of TGF-alpha and EGFR by tumor cells is thought to trigger the autocrine growth pathway, leading to uncontrolled proliferation. To examine their roles in rat esophageal tumorigenesis induced by the chemical carcinogen N-nitrosomethylbenzylamine (NMBA), TGF-alpha, and EGFR expression was evaluated in normal rat esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical analyses. Compared with the levels in normal epithelium, the TGF-alpha and EGFR mRNA levels in esophageal papillomas were 3.6 and 1.9 times higher, respectively. In the preneoplastic epithelium, although a trend of increased TGF-alpha and EGFR mRNA levels was observed, collectively there were no significant differences between preneoplastic and normal samples by RT-PCR analysis. In situ hybridization and immunohistochemical staining showed increased levels of TGF-alpha and EGFR mRNA and protein products in papillomas and in pronounced hyperplastic and dysplastic lesions. TGF-alpha and EGFR expression correlated with each other and with the expression of proliferating cell nuclear antigen, a marker for cell proliferation. These results suggest that disregulation of TGF-alpha and EGFR expression may contribute to autonomous cell growth and may play an important role in rat esophageal tumorigenesis induced by NMBA.

Granulocyte-macrophage colony stimulating factor gene expression and function during tumor promotion.

Although recent evidence suggests that granulocyte-macrophage colony stimulating factor (GM-CSF) plays a role in cutaneous inflammation induced by topical exposure of phorbol ester tumor promoters to murine epidermis, there is little information available on the temporal sequence of gene expression of this cytokine over the time course of tumor promotion or about its function in this process. The goal of the present studies was to examine the potential role of GM-CSF in tumor promotion in SENCAR mice. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) studies demonstrated that a single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA; 2 micrograms, 10 micrograms) to the dorsal epidermis of SENCAR mouse skin stimulated a dose and time dependent GM-CSF gene expression that was upregulated at 1 h after TPA exposure, peaked at 3 h and declined at 12 h. Although treatment with 7',12'-dimethylbenz[a]anthracene (DMBA) did not stimulate GM-CSF gene expression, GM-CSF gene expression was elevated in epidermal tissue isolated from SENCAR mice treated with a single application of 10 nmol DMBA followed by multiple applications of 2 micrograms TPA over a 1-22 week time course. Immunochemical and autoradiographic studies demonstrated that GM-CSF protein was produced by suprabasal keratinocytes, interfollicular cells, nonproliferating papilloma cells and leukocytes within the dermis. Intraperitoneal injection of recombinant (r) GM-CSF into SENCAR mice at 2 h prior to topical application of 10 micrograms TPA induced a significant increase in epidermal keratinocyte proliferation, leukocyte infiltration into the dermis, hydroperoxide production by circulating neutrophils and chemotactic activity present within the plasma at 24 h compared to treatment with only 10 micrograms TPA. Intravenous injection of anti-GM-CSF antibodies significantly inhibited both local and systemic inflammatory events induced by topical application of TPA. The present studies suggest that GM-CSF has a broad spectrum of activity with at least two target cell populations, epidermal keratinocytes within the proliferative compartment and leukocytes. This cytokine is actively transcribed during the tumor promotion process, acts as a signal peptide that stimulates epidermal proliferation, primes circulating neutrophils to produce hydroperoxide and regulates leukocyte migration.

Interleukin-1alpha gene expression during wound healing.

Interleukin-1alpha is known to be constitutively produced by epidermal keratinocytes under normal conditions, and injection of this cytokine enhances wound reepithelialization. However, no studies have characterized the temporal sequence of interleukin-1alpha gene expression over the time course of wound healing, and the cellular sources of this cytokine have not been identified. In the present studies, levels of interleukin-1alpha messenger RNA in wound tissue isolated from SKH-1 hairless mice were characterized and the cells that produced interleukin-1alpha immunoreactive protein over a 10-day time course of wound healing were defined. A time-dependent upregulation in interleukin-1alpha gene expression occurred immediately (4 hours) after a full-thickness wound was made, which represented a four-fold increase over levels of cytokine gene expression detected in nonwounded skin. Upregulation of cytokine gene expression correlated with an immediate increase in plasma interleukin-1alpha levels and was followed by an increase in interleukin-1alpha immunoreactive protein localized to keratinocytes within the leading edge of the wound and epidermis, as well as to neutrophils within the dermis. The rapid increase in local and systemic interleukin-1alpha levels correlated with the infiltration of a significant number of neutrophils into the wound site and with the proliferation of both basal keratinocytes and dermal fibroblasts. Given the known ability of interleukin-1alpha to regulate proliferation and migration of epidermal keratinocytes and to indirectly induce leukocyte chemotaxis, the results of the present studies suggest that interleukin-1alpha may be an important cytokine with both local and systemic actions that are linked to the initiation of critical cellular events early in wound healing.