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CYP1A1 and CYP1A2 are transcriptionally activated in the human normal breast epithelial cell line MCF10A following exposure to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Shifting MCF10A cultures to medium deficient in serum and epidermal growth factor (EGF) caused rapid reductions in the activated (i.e., phosphorylated) forms of extracellular regulated kinases (ERKs) and the epidermal growth factor receptor (EGFR). Shifting to serum/EGF-deficient medium also enhanced TCDD-mediated induction of cytochrome P450 ( CYP ) 1A1 . Treatment of cells cultured in complete medium with the EGFR inhibitors gefitinib (Iressa), AG1478, and CI-1033 resulted in concentration-dependent reductions of active EGFR and ERKs, and increased CYP1A1 mRNA content ∼3- to 18-fold above basal level. EGFR inhibitors synergized with TCDD and resulted in transient CYP1A1 and CYP1A2 mRNA accumulations ∼8-fold greater (maximum at 5 hours) than that achieved with only TCDD. AG1478, gefitinib, and TCDD individually induced small increases (∼1.2- to 2.5-fold) in CYP1A1 protein content but did not cause additive or synergistic accumulations of CYP1A1 protein when used in combination. The mitogen-activated protein kinase kinase inhibitor PD184352 inhibited ERK and EGFR activation in a concentration-dependent fashion without causing CYP1A1 mRNA accumulation. However, cotreatment with PD184352 potentiated TCDD-mediated CYP1A1 induction. TCDD-mediated induction of CYP1A1 in MCF7-TETon-EGFR cells, a MCF7 variant in which EGFR expression can be controlled, was not affected by the activity status of EGFR or ERKs. Hence, EGFR signaling mutes both basal and ligand-induced expression of two aryl hydrocarbon receptor–responsive P450s in MCF10A cultures. However, these effects are cell context–dependent. Furthermore, CYP1A1 mRNA and protein abundance are not closely coupled in MCF10A cultures.

Detroit, Michigan, currently has the highest preterm birth (PTB) rate of large cities in the United States. Disproportionate exposure to ambient air pollutants, including particulate matter ≤2.5 μm (PM2.5), PM ≤ 10 μm (PM10), nitrogen dioxide (NO2) and benzene, toluene, ethylbenzene, and xylenes (BTEX) may contribute to PTB. Our objective was to examine the association of airshed pollutants with PTB in Detroit, MI. The Geospatial Determinants of Health Outcomes Consortium (GeoDHOC) study collected air pollution measurements at 68 sites in Detroit in September 2008 and June 2009. GeoDHOC data were coupled with 2008–2010 Michigan Air Sampling Network measurements in Detroit to develop monthly ambient air pollution estimates at a spatial density of 300 m2. Using delivery records from two urban hospitals, we established a retrospective birth cohort of births by Detroit women occurring from June 2008 to May 2010. Estimates of air pollutant exposure throughout pregnancy were assigned to maternal address at delivery. Our analytic sample size included 7961 births; 891 (11.2%) were PTB. After covariate adjustment, PM10 (P = 0.003) and BTEX (P

We previously reported that low level lysosomal photodamage enhanced the efficacy of subsequent mitochondrial photodamage, resulting in a substantial promotion of apoptotic cell death. We now extend our analysis of the sequential PDT protocol to include two additional lysosomal-targeting photosensitizers. These agents, because of enhanced permeability, are more potent than the agent (N-aspartyl chlorin E6, NPe6) used in the initial study. Addition of the cell-permeable cysteine protease inhibitor E-64d and calcium chelator BAPTA-AM almost completely suppressed sequential PDT-induced loss of mitochondrial membrane potential, activation of procaspases-3 and -7 and loss of colony formation. These inhibitors did not, however, suppress the pro-apoptotic effect of a BH3 mimetic or mitochondrial photodamage. Knockdowns of ATG7 or ATG5, proteins normally associated with autophagy, suppressed photodamage induced by the sequential PDT protocol. These effects appear to be independent of the autophagic process since pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdown may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial pro-apoptotic processes. Our results suggest that low-dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers.

Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin.

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD(50) light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.

Dept. of Civil and Environmental Engineering, University of Windsor, Windsor, Ontario, Canada Dept. of Geology, Wayne State University, 0224 Old Main, 4841 Cass, Detroit, MI 48202, USA Air Health Science Division, Health Canada, Ottawa, Ontario, Canada Dept. of Family Medicine, Wayne State University, Detroit, MI, USA Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario, Canada Henry Ford Health System, Detroit, MI, USA College of Nursing, Wayne State University, Detroit, MI, USA h School of Nursing, Eastern Michigan University, Ypsilanti, MI, USA i Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA

Exposure of the human malignant peripheral nerve sheath tumor cell lines STS-26T, ST88-14, and NF90-8 to nanomolar concentrations of both lovastatin and farnesyl transferase inhibitor (FTI)-1 but not to either drug alone induced cell death. ST88-14 and NF90-8 cells underwent apoptosis, yet dying STS-26T cells did not. FTI-1 cotreatment induced a strong and sustained autophagic response as indicated by analyses of microtubule-associated protein-1 light chain 3 (LC3)-II accumulation in STS-26T cultures. Extensive colocalization of LC3-positive punctate spots was observed with both lysosome-associated membrane protein (LAMP)-1 and LAMP-2 (markers of late endosomes/lysosomes) in solvent or FTI-1 or lovastatin-treated STS-26T cultures but very little colocalization in lovastatin/FTI-1-cotreated cultures. The absence of colocalization in the cotreatment protocol correlated with loss of LAMP-2 expression. Autophagic flux studies indicated that lovastatin/FTI-1 cotreatment inhibited the completion of the autophagic program. In contrast, rapamycin induced an autophagic response that was associated with cytostasis but maintenance of viability. These studies indicate that cotreatment of STS-26T cells with lovastatin and FTI-1 induces an abortive autophagic program and nonapoptotic cell death.

Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles, and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable non-esterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal non-esterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspases-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the inductions of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal non-esterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP, and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins.

As part of a larger research project initiated by the Geospatial Determinants of Health Outcomes Consortium (GeoDHOC), an air quality study was conducted in an international airshed encompassing Detroit, Michigan, USA, and Windsor, Ontario, Canada. Active and passive samplers were used to measure nitrogen dioxide (NO2), sulfur dioxide (SO2), 26 volatile organic compounds (VOCs), 23 polycyclic aromatic hydrocarbons (PAHs) and pesticides, and three size fractions of particulate matter (PM) over a two-week period in September, 2008. Measurements of NO2 and 14 VOCs were found to be acceptable at 98 out of 100 passive monitoring sites. PAH and PM measurements were acceptable at 38 out of 50 active sites. Mean concentrations for all analytes except for PM2.5–10 were higher in Detroit than Windsor by a factor of up to 1.8. Strong statistical correlations were found among benzene, toluene, ethylbenzene, and xylene (BTEX), as well as between NO2 and PM in Detroit. In Windsor, the strongest correlations were between NO2 and total VOCs, as well as total PAHs and total VOCs. Differences in the degree of correlation observed in Detroit and Windsor are attributable to differences in the volume and composition of emissions within the two cities. Spatial variability was evaluated using a combination of statistical (coefficient of variation) and geostatistical (standardized variogram slope) metrics together with concentration maps. Greater spatial variability was observed for total VOCs and total BTEX in Detroit, while greater variability of NO2, total PAHs, and PM was found in Windsor. Results of this study suggest that statistical correlations between NO2 and other contaminants may not provide sufficient justification for the indiscriminant use of NO2 as a proxy for those contaminants if smaller scale features are to be reproduced.

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G0/G1 arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.

The therapeutic usefulness of the quinoxaline derivatives XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid) has been attributed to their abilities to induce G2/M arrest and apoptotic or autophagic cell death. Concentrations of XK469 or SH80 ≥ 5 μM were cytostatic to cultures of the normal murine melanocyte cell line Melan-a. Higher concentrations caused dose-dependent cytotoxicity. Concentrations ≥10 μM provoked dramatic morphological changes typified by marked increases in cell size and granularity. XK469/SH80-treated cultures accumulated tetraploid (4N) DNA-containing cells within 24 h of treatment, an 8N population within 3 days, and a 16N population within 5 days. Increases in ploidy correlated with the appearance of multinucleated cells. Under no circumstances did cells exhibit evidence of furrow formation. Both drugs suppressed cytokinesis in additional mammalian cell lines. Cytotoxic concentrations of XK469 elevated DEVDase activities (a measure of procaspase-3/7 activation) and enhanced cellular staining by a fluorescent analog of the pan caspase inhibitor valine-alanine-aspartic acid-fluoromethyl ketone within 48 to 96 h of treatment. Within 48 h of treatment, cytostatic and cytotoxic concentrations of XK469 elevated p21 contents, reduced Bcl-2 and Bcl-XL contents, and induced autophagy, as monitored by the accumulaton of phosphatidylethanolamine-modified cleavage product of microtubule-associated protein light chain 3 (LC3-II). Cultures treated with ≥10 μM XK469 or SH80 for 5 days could not be induced to divide upon removal of drugs. Such cultures maintained high LC3-II contents, exhibited reduced cyclin E and D1 contents, and extensively expressed senescence-associated β-galactosidase within 14 to 17 days of cessation of drug treatment. Hence, XK469 and SH80 inhibit cytokinesis, promote polyploidy, and induce senescence in Melan-a cells.

Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain malignancies, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable, and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases, and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2, and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.

The proapoptotic effects of the Bcl-2 antagonist HA14-1 are believed to derive from its affinity for the hydrophobic groove on Bcl-2 and Bcl-x(L), thereby displacing proapoptotic factors, e.g. Bax and Bak. We have reported that HA14-1 promotes the efficacy of low-dose photodynamic therapy (PDT). A recent report proposed that the proapoptotic activity of HA14-1 reflects its ability to generate reactive oxygen species (ROS) when incubated in an aqueous environment. This later study, like several other HA14-1 investigations, relied on the use of fluorescent probes for ROS detection. We found that HA14-1 reacts with the albumin in serum to yield a fluorescent product. After correcting for this effect, the putative formation of ROS by HA14-1 could not be demonstrated with the fluorescent probes H(2)DCFDA, dihydroethidium or dihydrorhodamine. Indeed, the fluorescence excitation/emission spectra of HA14-1 encompassed the excitation/emission wavelengths used to detect these ROS probes. Cells cultured in a medium supplemented with ovalbumin, instead of serum, underwent apoptosis following HA14-1 addition, but did not exhibit fluorescence. Hence, HA14-1 fluorescence was unrelated to its proapoptotic activity. We conclude that the enhancement of PDT by HA14-1 reflects a pharmacologic effect, rather than its direct contribution of ROS.

Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G(1) DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.

Oncogenic Ras proteins are a driving force in a significant set of human cancers and wild-type, unmutated Ras proteins likely contribute to the malignant phenotype of many more. The overall challenge of targeting activated Ras proteins has great promise to treat cancer, but this goal has yet to be achieved. Significant efforts and resources have been committed to inhibiting Ras, but these energies have so far made little impact in the clinic. Direct attempts to target activated Ras proteins have faced many obstacles, including the fundamental nature of the gain-of-function oncogenic activity being produced by a loss-of-function at the biochemical level. Nevertheless, there has been very promising recent pre-clinical progress. The major strategy that has so far reached the clinic aimed to inhibit activated Ras indirectly through blocking its post-translational modification and inducing its mislocalization. While these efforts to indirectly target Ras through inhibition of farnesyl transferase (FTase) were rationally designed, this strategy suffered from insufficient attention to the distinctions between the isoforms of Ras. This led to subsequent failures in large-scale clinical trials targeting K-Ras driven lung, colon, and pancreatic cancers. Despite these setbacks, efforts to indirectly target activated Ras through inducing its mislocalization have persisted. It is plausible that FTase inhibitors may still have some utility in the clinic, perhaps in combination with statins or other agents. Alternative approaches for inducing mislocalization of Ras through disruption of its palmitoylation cycle or interaction with chaperone proteins are in early stages of development.

Photodynamic therapy (PDT) can cause lethal photodamage by both direct and indirect mechanisms. Direct modes of cell death relate to nonspecific necrosis and the initiation of signaling pathways that elicit apoptosis, autophagy or both. In this report, effects of low-dose and high-dose PDT are explored, comparing sensitizers that localize in the endoplasmic reticulum (the porphycene termed CPO) or mitochondria (mesochlorin). To explore the role of autophagy, two cell lines were examined-the murine L1210 leukemia and an Atg7 knockdown derivative of L1210. The Atg7 gene is central to the process of autophagy. High-dose PDT with either sensitizer resulted in a substantial loss of the Bcl-2 protein. As Bcl-2 regulates both apoptosis and autophagy, loss of this protein can lead to initiation of either or both processes. Low-dose PDT with either sensitizer resulted in the initiation of apoptosis in the L1210/Atg7 - cell line and a 20% loss of viability. In contrast, the same PDT dose led to the rapid appearance of autophagic cells in the L1210 line, less apoptosis and only a 5% loss of viability. These results are consistent with autophagy serving as a pro-survival response via the recycling of damaged organelles. At a higher PDT dose more apoptosis was again seen in the L1210/Atg7 - line, but both cell lines exhibited comparable cytotoxicity in colony formation assays. We conclude that autophagy offers protection from the phototoxic effects of low-dose PDT, but can serve as an alternate death mode when the PDT dose is increased.

In this study, murine leukemia L1210 cells were used to compare the effects of photodynamic therapy (PDT) with those of the apoptotic nonpeptidic Bcl-2 ligand ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1). The photosensitizing agent capronyloxy-tetrakis methyloxyethyl porphycene (CPO) was selected from a group of sensitizers previously shown to target the antiapoptotic protein Bcl-2 for photodamage. Like PDT with CPO, HA14-1 caused the rapid activation of procaspase-3, followed by the appearance of an apoptotic morphology within 60 min. Caspase activation after a sublethal dose of either PDT or HA14-1 was enhanced by staurosporine or the bile acid ursodeoxycholic acid. Moreover, PDT promoted procaspase activation and lethality of HA14-1 and vice versa. Effects of PDT versus HA14-1 could not be distinguished on the basis of the reactive oxygen species formation. Both caused the rapid oxidation of 2′,7′-dichlorofluorescein. These results are consistent wit...

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome. NF1 patients present with a constellation of clinical manifestations and have an increased risk of developing certain benign and malignant tumors. This disease results from mutation within the gene encoding neurofibromin, a GTPase activating protein (GAP) for Ras. Functional loss of this protein compromises Ras inactivation, which leads to the aberrant growth and proliferation of neural crest-derived cells and, ultimately, tumor formation. Current management of NF1-associated malignancy involves radiation, surgical excision, and cytotoxic drugs. The limited success of these strategies has fueled researchers to further elucidate the molecular changes that drive tumor formation and progression. This discussion will highlight how intracellular signaling molecules, cell-surface receptors, and the tumor microenvironment constitute potential therapeutic targets, which may be relevant not only to NF1-related malignancy but also to other human cancers.

Recent studies suggest that the aryl hydrocarbon receptor (AhR) modulates susceptibilities to some pro-apoptotic agents. AhR-containing murine hepatoma 1c1c7 cultures underwent apoptosis following exposure to tumor necrosis factor-alpha (TNFalpha) + cycloheximide (CHX). In contrast, Tao cells, an AhR-deficient variant of the 1c1c7 line, were refractory to this treatment. AhR sense/antisense transfection studies demonstrated that AhR contents influenced susceptibility to the pro-apoptotic effects of TNFalpha + CHX. 1c1c7 cells and all variants expressed comparable amounts of TNF receptor-1 and TRADD. However, no cell line expressed FADD, and consequently pro-caspase-8 was not activated. AhR content did not influence JNK and NF-kappaB activation. However, Bid and pro-caspase-9, -3, and -12 processing occurred only in AhR-containing cells. Analyses of cathepsin B and D activities in digitonin-permeabilized cultures and the monitoring of cathepsin B/D co-localization with Lamp-1 indicated that TNFalpha + CHX disrupted late endosomes/lysosomes in only AhR-containing cells. Stabilization of acidic organelles with 3-O-methylsphingomyelin inhibited TNFalpha + CHX-induced apoptosis. The cathepsin D inhibitor pepstatin A suppressed in vitro cleavage of Bid by 1c1c7 lysosomal extracts. It also delayed the induction of apoptosis and partially prevented Bid cleavage and the activation of pro-caspases-3/7 in cultures treated with TNFalpha + CHX. Similar suppressive effects occurred in cultures transfected with murine Bid antisense oligonucleotides. These studies showed that in cells where pro-caspase-8 is not activated, TNFalpha + CHX can initiate apoptosis through lysosomal disruption. Released proteases such as cathepsin D trigger the apoptotic program by activating Bid. Furthermore, in the absence of exogenous ligand, the AhR modulates lysosomal disruption/permeability.

Type 1 neurofibromatosis (NF1) is a common autosomal dominant disorder that results in neuroectodermal tumors. The NF1 tumor-suppressor gene encodes neurofibromin, which includes a GTPase-activating domain for Ras inactivation. Affinity purification showed N-Ras to be the predominant activated isoform of Ras in two independent neurofibrosarcoma cell lines from NF1 patients (lines ST88-14 and NF90-8). These NF1 cells also demonstrated increased constitutive activity of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) mitogen-activated protein (MAP) kinases compared with a sporadic malignant schwannoma cell line that maintains neurofibromin expression (STS-26T). Thus, MAP kinase kinase (MEK) inhibitors may be a rational approach to NF1 therapy. The MEK inhibitors PD98059 [2′-amino-3′-methoxyflavone], PD184352 (also called CI-1040) [2-(2-chloro-4-iodo-phenylamino)- N -cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] all produced concentration-dependent suppression of the proliferation of the three cell lines. Individual MEK inhibitors had similar effects in all three cell lines. However, only the antiproliferative effects of PD184352 correlated closely with the elimination of ERK1,2 MAP kinase activities. PD98059 was primarily cytostatic, whereas U0126 and PD184352 were cytotoxic. Only PD184352 induced apoptosis in all three lines, as indicated by morphology, activation of DEVDase, procaspase-3 cleavage, and the appearance of populations having sub-G 0 /G 1 DNA contents. The differential effects of the MEK inhibitors on cell survival were not dependent on p53 status or effects on the ERK5 pathway. PD184352 was also proapoptotic to primary rat Schwann cells. Hence, although PD184352 effectively killed neurofibrosarcoma cells, its effects on normal Schwann cells may limit its usefulness in the clinic.

Irradiation of murine hepatoma 1c1c7 cultures presensitized with N-aspartyl chlorin e6 (NPe6) caused lysosomal disruption and apoptosis. Tao cells, a variant of the 1c1c7 line having lower aryl hydrocarbon receptor (AhR) contents, were resistant to the pro-apoptotic effects of NPe6 in the same photodynamic therapy protocol. Colony-forming assays were used to establish light dose-dependent and NPe6 concentration-dependent cytotoxicity curves. Lysosomal breakage and cell survival paralleled one another in both cell types. When analyzed at comparable lethal dose conditions, the onset of apoptosis was delayed, and the magnitude of the apoptotic response was muted in Tao cells, as assessed by morphology, annexin V binding, caspase-3 activities, and analyses of Bid, procaspase-9, and pro-caspase-3 cleavage. In contrast, the kinetics/magnitude of pro-caspase-3 activation in the two cell lines were identical after exposure to HA14 -1 or Jo2 antibody, inducers of the intrinsic and extrinsic apoptotic pathways, respectively. Tao endosomal/lysosomal extracts contained approximately 50%, 35%, and 55% of the Bid cleavage and cathepsin B and D activities of 1c1c7 endosomes/lysosomes, respectively. Western blot analyses confirmed reduced cathepsin B/D contents in Tao cells. Analyses of 1c1c7/Tao variants engineered to express antisense/sense AhR constructs suggested that endosomal/lysosomal cathepsin B and D content, but not whole cell content, correlated with AhR expression. These studies provide a mechanism for the resistance of Tao cultures to the proapoptotic effects of a protocol causing targeted disruption of lysosomes. They also suggest that the AhR, in the absence of exogenous ligand, may affect the trafficking/processing of proteases normally found in endosomes/lysosomes.

Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA reductase and thus the synthesis of cholesterol, are remarkably effective in the treatment of cardiovascular disease. In addition to their favorable effect on lipid profile, these drugs may also prevent the proliferation of vascular smooth muscle that is characteristic of atherosclerosis. We hypothesize that statins prevent the post-translational prenylation, and thus inhibit the function, of critical small GTPases in vascular smooth muscle cells. We have therefore assayed the effect of lovastatin on both the growth of A10 vascular smooth muscle cells and the status of their Ras and RhoB proteins. We find thator =1 microM lovastatin potently inhibits the proliferation of A10 cultures, and higher concentrations (or =3 microM) induce apoptosis. We have also tested the effect of 3-allylfarnesol (3-alFOH), an inhibitor of farnesyl transferase (FTI). The data show that althoughor =10 microM 3-alFOH is required for a cytostatic effect, the action of 3 microM 3-alFOH can be greatly potentiated by even nanomolar levels of lovastatin. We also find that lovastatin and 3-alFOH exhibit synergism to cause the up-regulation and relocalization of RhoB from the membrane to cytosolic compartments. This relocalization of RhoB, which is presumed to reflect an inhibition of its prenylation, correlates with the proapoptotic activities of combined 3-alFOH and lovastatin treatment. These data suggest that RhoB may be a valuable pharmacological target in cardiovascular disease, and that combinations of statins and certain FTIs may be of value in treatment of disorders that are characterized by excess cell proliferation.

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-β in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and β-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-β antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-β.

This study was designed to examine determinants of the discovery that low-dose lysosomal photodamage (lyso-PDT) could potentiate the efficacy of subsequent low-dose mitochondrial photodamage (mito-PDT). The chlorin NPe6 and the benzoporphyrin derivative (BPD) were used to separately target lysosomes and mitochondria, respectively, in murine hepatoma cells. Lyso-PDT (LD(5) conditions) followed by mito-PDT (LD(15) conditions) enhanced the loss of the mitochondrial membrane potential, activation of procaspases-3/7 and photokilling. Reversing the sequence was less effective. The optimal sequence did not enhance reactive oxygen species formation above that obtained with low-dose mito-PDT. In contrast, alkalinization of lysosomes with bafilomycin also enhanced low-dose mito-PDT photokilling, but via a different pathway. This involves redistribution of iron from lysosomes to mitochondria leading to enhanced hydroxyl radical formation, effects not observed after the sequential procedure. Moreover, Ru360, an inhibitor of mitochondrial calcium and iron uptake, partially suppressed the ability of bafilomycin to enhance mito-PDT photokilling without affecting the enhanced efficacy of the sequential protocol. We conclude that sequential PDT protocol promotes PDT efficacy by a process not involving iron translocation, but via promotion of the pro-apoptotic signal that derives from mitochondrial photodamage.

While some epidemiological risk factors for breast cancer have been identified, the environmental factors responsible for transformation of mammary epithelial cells are not clear. We have exposed the spontaneously immortalized human mammary epithelial cell line MCF-10A to benzo[a]pyrene and selected transformed clones based on a loss of contact inhibition and anchorage-dependent growth. Cytogenetic studies showed that each of the transformed sublines possess an isochromosome 8q aberration. The c-Myc proto-oncogene, which is positioned at 8q24, was analyzed for changes in expression. Both c-Myc mRNA and protein levels were increased in the transformed clones relative to the parental cells. The transformed clones were not able to grow as tumors in vivo when injected into nude or SCID mice. To determine whether the involvement of chromosome 8 in BP-induced mutagenesis was a reproducible event, transformed clones were selected from three additional independently treated sets of BP-exposed MCF-10A cultures and analyzed by spectral karyotyping (SKY). These transformed sublines also harbored the isochromosome 8q abnormality. Data from this model show that benzo[a]pyrene, a ubiquitous procarcinogen, can induce selectable morphologic changes in a human mammary epithelial cell line, and that these transformed cells possess chromosomal aberrations frequently found in human breast tumors.

Numerous flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and function as AHR antagonists and/or agonists. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] is a widely used inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), and is structurally related to members of the flavonoid family. Concentrations of LY294002/= 10 microM were cytostatic, but not cytotoxic, to cultures of the immortalized human breast epithelial cell line MCF10A-Neo. Treatment of MCF10A-Neo cultures with the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of CYP1A1, as monitored by measurements of steady-state CYP1A1 mRNA. Pretreatment of cultures with/= 10 microM LY294002 suppressed the TCDD activation of CYP1A1 (IC(50) approximately 10 microM). Electrophoretic mobility shift assays employing rat liver cytosol demonstrated that concentrations of LY294002/= 400 microM did not transform the AHR into a DNA-binding species. However, the addition of LY294002 to cytosol just prior to TCDD addition completely suppressed AHR transformation by TCDD (IC(50) approximately 35 microM). The PI 3-kinase inhibitor Wortmannin was weakly cytostatic, but not cytotoxic to MCF10A-Neo cultures at concentrations/= 500 nM. Exposure of cultures to Wortmannin (10-500 nM) did not suppress TCDD activation of CYP1A1. Analyses of the phosphorylation status of Akt-1, an in vivo substrate of PI 3-kinase, demonstrated that concentrations of LY294002/= 50 microM and Wortmannin/= 10 nM completely suppressed PI 3-kinase activity. Hence, the ability of LY294002 to suppress TCDD-dependent activation of CYP1A1 is unrelated to PI 3-kinase inhibition. Instead, this activity reflects LY294002 functioning as an AHR antagonist. Furthermore, most of the cytostatic activity of LY294002 towards MCF10A-Neo cells is unrelated to the inhibition of PI 3-kinase.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for

The effect of oral administration of lead, as Pb-acetate, via the drinking water on the murine delayed type hypersensitivity (DTH) response was investigated. The DTH response of BALB/c mice sensitized intraveneously with sheep red blood cells (SRBC) was found to be suppressed markedly in lead-intoxicated mice. Suppression of the DTH correlated with increasing blood Pb concentration. Suppression of the DTH response by Pb intoxication depended on the route of administration of the sensitizing antigen, as Pb intoxication did not impair the DTH reaction when mice were sensitized to SRBC via intraperitoneal injection. Since DTH reactions are regulated in large part by Th1 cells, these data establish an in vivo model system based on a rational route of Pb exposure in which to study further the modulation of Th1-mediated immune effector function by Pb.

The relationship between aryl hydrocarbon receptor (AHR) content and susceptibility to apoptosis was examined in the murine hepatoma 1c1c7 cell line and a series of variants having different levels of AHR expression. Exposure of 1c1c7 cultures toN-acetylsphingosine (C2-ceramide) caused a concentration-dependent inhibition of cell proliferation, loss of viability, and induction of apoptosis as monitored by analyses of DNA fragmentation and caspase activation. A variant cell line (Tao) having ∼10% of the AHR content of 1c1c7 cells also arrested following exposure to C2-ceramide, but did not undergo apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection ofAhr antisense and sense constructs, respectively, confirmed the relationship between AHR content and susceptibility to C2-ceramide-induced apoptosis. C2-ceramide also induced the apoptosis of an AHR-containing cell line lacking the aryl hydrocarbon receptor nuclear translocator protein. AHR ligands (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone) neither induced apoptosis nor modulated the development of apoptosis in C2-ceramide-treated 1c1c7 cultures. AHR content did not affect staurosporine- or doxorubicin-induced apoptosis. These results suggest the AHR modulates aspects of ceramide signaling associated with the induction of apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear translocator and exogenous AHR ligands.

PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] is a flavonoid and a potent inhibitor of mitogen-activated protein kinase kinase (MEK). Concentrations of PD98059 of/=20 muM were not cytotoxic to cultures of the immortalized human breast epithelial cell line MCF10A. The agent was weakly cytostatic at concentrations of/=10 microM. In vivo exposure of cultures to/=20 microM PD98059 for 2-22 hr did not affect overall extracellular signal-regulated kinase contents; however, exposure to PD98059 resulted in a rapid loss (95%) of the dually phosphorylated forms of extracellular signal-regulated kinase (IC50 = 1 muM). Treatment of cultures with PD98059 of/=1 muM either at the time of addition or up to 48 hr before the addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed in a concentration-dependent manner the accumulation of induced steady state CYP1A1, CYP1B1, and NQO1 mRNAs. The addition of PD98059 to rat liver cytosol just before the addition of TCDD suppressed TCDD binding (IC50 = 4 muM) and aryl hydrocarbon receptor (AHR) transformation (IC50 = 1 muM), as measured by sucrose gradient centrifugation and electrophoretic mobility shift assays. Flavone and flavanone, two closely related structural analogs of PD98059, inhibited AHR transformation by TCDD with IC50 values similar to that obtained with PD98059. However, neither analog was as potent as PD98059 in inhibiting MEK (IC50 approximately 190 muM for both). These results suggest that PD98059 is a ligand for the AHR and functions as an AHR antagonist at concentrations commonly used to inhibit MEK and signaling processes that entail MEK activation.

Products of several phase I and II genes transcriptionally activated by ligands of the aryl hydrocarbon receptor (AHR) were quantitated in cutaneous samples isolated from non-tumor-bearing SENCAR or SSIN mice, and animals bearing skin tumors generated in initiation-promotion protocols. The constitutive 7-ethoxyresorufin O-deethylase (EROD) activities in papillomas and squamous cell carcinomas were less than or equal to 37% of the values measured in the adjacent normal cutaneoustissue. Dermal and epidermal EROD specific activities in microsomal samples prepared from both tumor-bearing and non-tumor-bearing mice were elevated 9- to 14- and 43- to 77-fold, respectively, above constitutive levels 16-20 h after a single topical application of 100 nmol of dibenz[a,c]anthracene (DB[a,c]A). EROD specific activities in tumors were maximally elevated two-fold after topical application of DB[a,c]A. Western blot, northern blot, and reverse transcription (RT)-polymerase chain reaction (PCR) analyses confirmed that the EROD measurements reflected cutaneous cytochrome P450 (CYP) 1A1 protein, mature mRNA, and heterogeneous nuclear RNA contents, respectively. Analyses of CYP1A1, CYP1B1, cytosolic aldehyde dehydrogenase class 3, and NAD(P)H:menadione oxidoreductase (NMO1) mRNA content by RT-PCR revealed significant increases in all four mRNAs in the normal tissue adjacent to papillomas after exposure to 4 nmol of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but no increases in the tumors. NMO1 mRNA content in acetone-treated papillomas approached the levels detected in TCDD-treated normal skin. RT-PCR analyses also demonstrated elevated constitutive aryl hydrocarbon receptor nuclear translocator mRNA content (an approximately two-fold increase) in skin tumors. In contrast, AHR mRNA content in the tumors was about 20% of that measured in adjacent normal tissue. Collectively, these studies demonstrated that ligand-induced, AHR-mediated processes are absent in murine skin tumors that develop in initiation-promotion protocols.

Primary murine keratinocytes were cultured in a chemically defined, serum-free medium which facilitated manipulation of their differentiation status. Exposure of basal cell and differentiating cultures to >/= 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) preferentially elevated 7-ethoxyresorufin O-deethylase specific activities in differentiating cultures (28-fold versus 4-fold increases after 36 h of exposure). Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses demonstrated the presence of constitutive mRNA transcripts corresponding to four known TCDD-inducible genes (e.g., Cyp1a1, Cyp1b1, Ahd4, and Nmo1) in both differentiating and proliferating cultures of murine keratinocytes. All four genes were induced in differentiating cultures following exposure to TCDD. No induction occurred in comparably treated basal cell cultures. Indirect immunofluorescence analyses demonstrated the presence of aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins in both basal and differentiating keratinocytes. Both proteins appeared to be associated with the nucleus and their nuclear association was independent of prior exposure to TCDD. These studies suggest that AHR activation in murine skin is regulated as a function of the keratinocyte differentiation program.

SSIN mice are very sensitive to tumor promoters in two-stage skin carcinogenesis protocols. It was recently reported that SSIN mice have fewer CD8+ T-cells than other strains of mice and develop a weaker cytotoxic T-cell response upon challenge with an allogeneic tumor transplant. The significance of this muted immune response to processes involved in two-stage carcinogenesis depends on the immunogenicity of the tumors generated in such protocols. Although they have low CD8+ T-cell contents, SSIN rejected a variety of subcutaneously transplanted allogeneic murine tumors. Analyses of the growth of primary papillomas derived from 7,12-dimethylbenz[a]anthracene–initiated/12-O-tetradecanoylphorbol-13-acetate–promoted SSIN mice and then subcutaneously transplanted into triple-deficient (bg-nu-xid), athymic nude and immune-competent and immunosuppressed SSIN mice revealed that few tumors took and tumor takes were not markedly influenced by the immumological status of the transplant recipient. Two tumor cell lines (RS1 and RS2) were derived from the transplantation studies and could be passaged in normal SSIN mice (H-2q haplotype). Both tumors were squamous cell carcinomas (SCCs) by the second in vivo passage and were rejected in allogeneic mice (BALB/c) but grew in FVB/N mice, a strain having the H-2q haplotype. Transplantation studies revealed that prior exposure to RS1 and RS2 did not prime SSIN mice to reject a subsequent tumor challenge. Three primary SCC tumors derived from SSIN mice in a two-stage carcinogenesis protocol also grew when subcutaneously transplanted in SSIN mice and could be serially passaged. Consequently, the epidermal SCCs that develop in two-stage carcinogenesis protocols appear to be nonimmunogenic. Mol. Carcinog. 20:48–57, 1997. © 1997 Wiley-Liss, Inc.

The immortalized human epithelial cell line MCF10A has the phenotypic characteristics of normal breast cells. Exposure of MCF10A cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of cytochrome P450 1A1 (CYP1A1), and CYP1B1, and NAD(P)H:quinone oxidoreductase. Northern blot hybridization and nuclear run-on assays demonstrated that transcriptional activation of these genes was suppressed in stably transfected cultures expressing an Ha-ras oncogene (the MCF10A-NeoT line). Similar suppression did not occur in stably transfected lines carrying the expression vector or a normal c-Ha-ras protooncogene. Western blot analyses and immunofluorescence microscopy demonstrated that the lack of inducibility in MDF10A-NeoT cells reflected neither reductions in aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator protein nor prevention of TCDD-induced AHR translocation to the nucleus. Suppression did correlate with reductions in DNA-AHR complex formation, as analyzed by gel retardation assays of soluble cell extracts treated in vitro with TCDD. The induction of Cyp1a-1 by TCDD was also analyzed in transgenic mice that expressed a v-Ha-ras oncogene exclusively in their keratinocytes. Relative to littermates lacking the transgene, the induction of Cyp1a-1 by TCDD was partially suppressed (about 50%) in the epidermises of v-Ha-ras-positive transgenic mice. However, normal levels of Cyp1a-1 induction occurred in the livers of the same mice. induction of Cyp1a-1 by TCDD was also suppressed (more than 98%) in chemically induced skin papillomas having Ha-ras mutations, relative to uninvolved surrounding skin. These studies suggest that the p21-ras protein controls signal transduction pathways capable of modulating AHR function.

The Geospatial Determinants of Health Outcomes Consortium (GeoDHOC) study investigated ambient air quality across the international border between Detroit, Michigan, USA and Windsor, Ontario, Canada and its association with acute asthma events in 5- to 89-year-old residents of these cities. NO2, SO2, and volatile organic compounds (VOCs) were measured at 100 sites, and particulate matter (PM) and polycyclic aromatic hydrocarbons (PAHs) at 50 sites during two 2-week sampling periods in 2008 and 2009. Acute asthma event rates across neighborhoods in each city were calculated using emergency room visits and hospitalizations and standardized to the overall age and gender distribution of the population in the two cities combined. Results demonstrate that intra-urban air quality variations are related to adverse respiratory events in both cities. Annual 2008 asthma rates exhibited statistically significant positive correlations with total VOCs and total benzene, toluene, ethylbenzene and xylene (BTEX) at 5-digit zip code scale spatial resolution in Detroit. In Windsor, NO2, VOCs, and PM10 concentrations correlated positively with 2008 asthma rates at a similar 3-digit postal forward sortation area scale. The study is limited by its coarse temporal resolution (comparing relatively short term air quality measurements to annual asthma health data) and interpretation of findings is complicated by contrasts in population demographics and health-care delivery systems in Detroit and Windsor.

The effects of topically applied 12-O-tetradecanoylphorbol-13-acetate (TPA), acetone, and ethanol on systemic immune function were analyzed in SSIN mice. A total of one, four, or eight dorsal applications of solvent (50-300 microl/treatment, 2x/week) affected neither the overall cellularity of the spleen nor the relative proportions of splenic cells expressing CD4, CD8, or Ig surface markers. In contrast, overall cellularities of the spleen increased and relative T cell content of the spleen decreased in mice treated multiple times with TPA (2 microg/application in 0.2 ml acetone). The development of splenic B cells secreting IgM against sheep red blood cells (SRBC, a T-cell-dependent antigen) was retarded, and the overall duration of IgM synthesis was decreased, in mice immunized 1 hr after the last of four applications (>200 microl) of either solvent. Comparable retardations occurred in mice immunized as late as 7 days after termination of solvent treatment. However, solvent effects on the development of antibody-forming cells were not observed after eight topical applications or when TNP-LPS (a T-cell-independent antigen) was used as the immunogen. The effects of TPA on the development of IgM-secreting B cells were indistinguishable from those of the solvent used for its application. Although serum hemagglutination titers to SRBC correlated with the relative numbers of splenic B cells producing IgM, in vitro proliferative responses to B and T cell mitogens were not predictive of the effects of solvents or TPA on the development of antibody-secreting cells. Collectively, these studies demonstrate that topically applied acetone and ethanol can systemically modulate humoral immunity and emphasize the need for inclusion of nontreated controls when assessing the potential immunomodulatory activities of agents dissolved in acetone or ethanol.

SSIN mice are considerably more sensitive to the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in two-stage skin carcinogenesis protocols than are most other strains and stocks of mice. Experiments were performed to determine whether there was an immunological basis for this sensitivity. SSIN mice were haplotyped and found to be H-2q. T cells represented approximately 31% of the splenic cellularity of non-treated SSIN mice, but approximately 44% in BALB/c, C57BL/6, B6C3F1 and SENCAR mice. Splenic CD4+/CD8+ T cell ratios were approximately 4.2, 2.9, 2.4, 1.8 and 1.7 in SSIN, SENCAR, BALB/c, B6C3F1 and C57BL/6 mice, respectively. The unusually high ratio in SSIN spleens was the consequence of reductions in CD8+ T cells. The ratio of CD4+/CD8+ T cells in SSIN thymocytes was similiar to that measured in the spleen. The splenic cytotoxic T lymphocyte (CTL) activities of the various murine strains inversely correlated with their splenic CD4+/CD8+ ratios and their sensitivities in two-stage skin carcinogenesis protocols. Repeated in vivo topical treatment of SSIN mice with TPA caused significant decreases in splenic T cell contents, but affected neither the splenic CD4+/CD8+ T cell ratio nor the development of a CTL response upon allogeneic tumor challenge. SSIN mice also had very low splenic natural killer cell activities. Furthermore, relative to the other strains of mice, SSIN mice were poor responders upon alloantigen challenge in mixed lymphocyte response assays. These findings demonstrate that SSIN mice differ markedly from other murine stocks and strains in their splenic lymphocyte composition and in their abilities to mount some MHC-restricted and non-restricted immunosurveillance processes.

Protein kinase C (PKC) activity is important in regulating cellular growth and differentiation and is believed to play a role in the promotional stage of carcinogenesis. To determine the effects of age and gender on PKC expression, isozyme-specific antibodies and immunoblot analyses were employed to examine the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon in the cytosolic and membrane fractions isolated from hepatic tissue of 1, 3, 5 and 12 week old male or female rats. PKC alpha levels were comparable at 1 week of age in the respective male and female hepatic fractions. In contrast, at 3, 5 and 12 weeks of age, cytosolic PKC alpha levels were approximately 2-, 5- and 7-fold greater, respectively, in females than in males. At 5 weeks of age, cytosolic levels of PKC delta were approximately 2-fold higher in females than in males. Other PKC isozymes were either below the limit of detection (PKC gamma), or failed to exhibit any gender-related differences (PKC beta and PKC epsilon). At 12 weeks of age, PKC activity was 1.7- or 2.4-fold greater in hepatic cytosol and membrane fractions, respectively, from females than in male samples. These results show distinct gender-specific and developmental differences in hepatic PKC isozyme expression, which may play a role in susceptibility to cancer.

Photodynamic therapy relies on the interaction between light, oxygen and a photosensitizing agent. Its medical significance relates to the ability of certain agents, usually based on porphyrin or phthalocyanine structures, to localize somewhat selectively in neoplastic cells and their vasculature. Subsequent irradiation, preferably at a sufficiently high wavelength to have a significant pathway through tissues, results in a photophysical reaction whereby the excited state of the photosensitizing agent transfers energy to molecular oxygen and results in the formation of reactive oxygen species. Analogous reactive nitrogen species are also formed. These contain both nitrogen and oxygen atoms. The net result is both direct tumor cell death and a shutdown of the tumor vasculature. Other processes may also occur that promote the anti-tumor response but these are outside the scope of this review.

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 μM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 μM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 μM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 μM pAA was cytostatic and induced a G(1) arrest that lasted ∼7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.

SENCAR mice develop more papillomas in two-stage skin carcinogenesis protocols if gamma interferon (IFN-gamma) is co-administered with 12-O-tetradecanoylphorbol-13-acetate (TPA) during the promotion phase. In the current study preparations of murine alpha, beta and gamma IFNs were surveyed for their abilities to modulate TPA-dependent promotion and induction of epidermal hyperplasia, inflammation and ornithine decarboxylase activity (ODC). Single or multiple i.p. administrations of IFN-alpha, -beta or -gamma (or = 2500 units) did not induce epidermal hyperplasia, inflammation or ODC activity. Single or multiple i.p. administrations of IFN-alpha, -beta or -gamma (2500 units) to mice being topically promoted with 0.1 or 1 microgram of TPA did not alter the epidermal hyperplasia induced by the phorbol ester. The vascular permeability of the skin, as evaluated by the extravasation of Evans blue dye, was increased in a dose-dependent fashion by TPA over the range of 0.1-1 microgram. Treatment of mice promoted with 0.1 microgram of TPA with IFN-gamma (or = 2500 units) significantly increased the skin's vascular permeability. Comparable effects were not obtained with IFN-beta (IFN-alpha not tested). Treatment of TPA-promoted mice with IFN-gamma, and to a lesser extent IFN-beta, weakly potentiated the TPA-dependent induction of epidermal ODC activity. Under conditions in which IFN-gamma had co-promoting activities in an initiation-promotion protocol, co-treatment of initiated mice with 1 microgram of TPA and IFN-alpha or -beta (100-5000 units) did not reproducibly alter tumor latency., or papilloma and carcinoma multiplicities. These findings suggest that the co-promoting activities of IFNs are restricted to the gamma class, and are not uniformly reflected by parameters commonly employed as short-term markers of tumor promotion.

Ear/footpad swelling following sensitization and challenge with 2,4-dinitrofluoro-benzene (DNFB)/allogenic splenocytes (AS) were used to monitor the effects of 12–0-tetradecanoylphorbol-13-acetate (TPA) on contact hypersensitivity (CHS) and delayed hypersensitivity (DHS) reactions, respectively. Topical treatment of dorsal or ventral SENCAR mouse skin 4x with 2 μg of TPA prior to sensitization of dorsal skin with DNFB suppressed attempts to induce CHS by susequent challenge with DNFB. the adoptive transfer of splenocytes isolated from mice pretreated on the dorsum with TPA prior to dorsal sensitization with DNFB inhibited the development of CHS to DNFB in recipient mice. Conversely, topical treatment with TPA prior to s.c. sensitization with AS neither suppressed subsequent attempts to induce DHS, nor resulted in the generation of a splenocyte population capable of suppressing DHS reactions in adoptive transfer studies. Thus, promoting doses of topically applied TPA has differential effects on CHS...

Plexiform neurofibromas commonly found in patients with Neurofibromatosis type I (NF1) have a 5% risk of being transformed into malignant peripheral nerve sheath tumors (MPNST). Germline mutations in the NF1 gene coding for neurofibromin, which is a Ras GTPase activating protein (RasGAP) and a negative regulator of Ras, result in an upregulation of the Ras pathway. We established a direct connection between neurofibromin deficiency and downstream effectors of Ras in cell lines from MPNST patients by demonstrating that knockdown of NF1 expression using siRNA in a NF1 wild type MPNST cell line, STS-26T, activates the Ras/ERK1,2 pathway and increases AP-1 binding and activity. We believe this is the first time the transactivation of AP-1 has been linked directly to neurofibromin deficiency in a disease relevant MPNST cell line. Previously, we have shown that N-Ras is constitutively activated in cell lines derived from independent MPNSTs from NF1 patients. We therefore sought to analyze the role of the N-Ras pathway in deregulating AP-1 transcriptional activity. We show that STS-26T clones conditionally expressing oncogenic N-Ras show increased phosphorylated ERK1,2 and phosphorylated JNK expression concomitant with increased AP-1 activity. MAP kinase pathways (ERK1,2 and JNK) were further examined in ST88-14, a neurofibromin-deficient MPNST cell line. The basal activity of ERK1,2 but not JNK was found to increase AP-1 activity. These experiments further confirmed the link between the loss of neurofibromin and increased activity of Ras/MAP kinase pathways and the activation of downstream transcriptional mechanisms in MPNSTs from NF1 patients.

Photodynamic therapy (PDT) is a procedure that has applications in the selective eradication of neoplasia where sites of malignant lesions are clearly delineated. It is a two-step process whereby cells are first sensitized to light and then photoirradiated. This results in the formation of singlet molecular oxygen and other reactive oxygen species that can cause photodamage at sites where the photosensitizing agent has localized. Photosensitizers found to be clinically useful show affinity for the endoplasmic reticulum (ER), mitochondria, lysosomes, or combinations of these sites. The induction of apoptosis and/or autophagy in photosensitized cells is a common outcome of PDT. This report explores the following issues: (1) Does the induction of autophagy in PDT protocols occur independent of, or in association with, apoptosis? (2) Does the resulting autophagy play a prosurvival or prodeath role? (3) Do photosensitizers damage/inactivate specific proteins that are components of, or that modulate the autophagic process? (4) Can an autophagic response be mounted in cells in which lysosomes are specifically photodamaged? In brief, autophagy can occur independently of apoptosis in PDT protocols, and appears to play a prosurvival role in apoptosis competent cells, and a prodeath role in apoptosis incompetent cells. Mitochondrial and ER-localized sensitizers cause selective photodamage to some (i.e., Bcl-2, Bcl-x(L), mTOR) proteins involved in the apoptotic/autophagic process. Finally, an aborted autophagic response occurs in cells with photodamaged lysosomes. Whereas autophagosomes form, digestion of their cargo is compromised because of the absence of functional lysosomes.

Epidermal 7-ethoxyresorufin O-deethylase (EROD) activity was elevated greater than 100-fold within 4 to 7 h of topical treatment of SENCAR mice with 100 nmol dibenz[a,c]anthracene (DB[a,c]A). Treatment of skin with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) 2 to 8 h prior to DB[a,c]A application suppressed induction by 80%. Suppression was dose-dependent over the range of 0.01 to 5 micrograms TPA (ID50 approximately 0.6 nmol). EROD activities in normal and TPA-treated epidermis paralleled steady state P450 CYP1A1 mRNA content. Analogs of TPA incapable of activating or down-regulating protein kinase C (PKC) did not suppress induction. Pretreatment of skin with sn-1,2-didecanoylglycerol, an activator of PKC which causes translocation but no down-regulation, did not suppress EROD induction. However, induction was suppressed by chrysarobin, an anthralin analog that causes PKC down-regulation in the absence of prior activation. These studies suggest that PKC participates in the processes associated with Cyp1a-1 induction and that TPA effects Cyp1a-1 induction through its down-regulation of PKC.