African American women in the United States have a high risk of adverse pregnancy outcomes. DNA methylation is a potential mechanism by which exposure to BTEX (benzene, toluene, ethylbenzene, and xylenes) may cause adverse pregnancy outcomes. Data are from the Maternal Stress Study, which recruited African American women in the second trimester of pregnancy from February 2009 to June 2010. DNA methylation was measured in archived DNA from venous blood collected in the second trimester. Trimester-specific exposure to airshed BTEX was estimated using maternal self-reported addresses and geospatial models of ambient air pollution developed as part of the Geospatial Determinants of Health Outcomes Consortium. Among the 64 women with exposure and outcome data available, 46 differentially methylated regions (DMRs) were associated with BTEX exposure (FDR adjusted p-value < 0.05) using a DMR-based epigenome-wide association study approach. Overall, 89% of DMRs consistently exhibited hypomethylation with increasing BTEX exposure. Biological pathway analysis identified 11 enriched pathways, with the top 3 involving gamma-aminobutyric acid receptor signaling, oxytocin in brain signaling, and the gustation pathway. These findings highlight the potential impact of BTEX on DNA methylation in pregnant women.

Neurofibromatosis type 1 (NF1) is a disorder in which RAS is constitutively activated due to the loss of the Ras-GTPase-activating activity of neurofibromin. RAS must be prenylated (i.e., farnesylated or geranylgeranylated) to traffic and function properly. Previous studies showed that the anti-growth properties of farnesyl monophosphate prodrug farnesyltransferase inhibitors (FTIs) on human NF1 malignant peripheral nerve sheath tumor (MPNST) cells are potentiated by co-treatment with lovastatin. Unfortunately, such prodrug FTIs have poor aqueous solubility. In this study, we synthesized a series of prodrug FTI polyamidoamine generation 4 (PAMAM G4) dendrimers that compete with farnesyl pyrophosphate for farnesyltransferase (Ftase) and assessed their effects on human NF1 MPNST S462TY cells. The prodrug 3-tert-butylfarnesyl monophosphate FTI-dendrimer (i.e., IG 2) exhibited improved aqueous solubility. Concentrations of IG 2 and lovastatin (as low as 0.1 μM) having little to no effect when used singularly synergistically suppressed cell proliferation, colony formation, and induced N-RAS, RAP1A, and RAB5A deprenylation when used in combination. Combinational treatment had no additive or synergistic effects on the proliferation/viability of immortalized normal rat Schwann cells, primary rat hepatocytes, or normal human mammary epithelial MCF10A cells. Combinational, but not singular, in vivo treatment markedly suppressed the growth of S462TY xenografts established in the sciatic nerves of immune-deficient mice. Hence, prodrug farnesyl monophosphate FTIs can be rendered water-soluble by conjugation to PAMAM G4 dendrimers and exhibit potent anti-tumor activity when combined with clinically achievable statin concentrations.

Air pollution is associated with preterm birth (PTB), potentially via inflammation. We recently showed the mixture benzene, toluene, ethylbenzene, and xylene (BTEX) is associated with PTB. We examined if ambient BTEX exposure is associated with mid-pregnancy inflammation in a sample of 140 African-American women residing in Detroit, Michigan. The Geospatial Determinants of Health Outcomes Consortium study collected outdoor air pollution measurements in Detroit; these data were coupled with Michigan Air Sampling Network measurements to develop monthly BTEX concentration estimates at a spatial density of 300 m2. First trimester and mid-pregnancy BTEX exposure estimates were assigned to maternal address. Mid-pregnancy (mean 21.3 +/- 3.7 weeks gestation) inflammatory biomarkers (high-sensitivity C-reactive protein, interleukin [IL]-6, IL10, IL-1 beta, and tumor necrosis factor-alpha) were measured with enzyme immunoassays. After covariate adjustment, for every 1-unit increase in first trimester BTEX, there was an expected mean increase in log-transformed IL-1 beta of 0.05 +/- 0.02 units (P = 0.014) and an expected mean increase in log-transformed tumor necrosis factor-alpha of 0.07 +/- 0.02 units (P = 0.006). Similarly, for every 1-unit increase in mid-pregnancy BTEX, there was a mean increase in log IL-1 beta of 0.06 +/- 0.03 units (P = 0.027). There was no association of either first trimester or midpregnancy BTEX with high-sensitivity C-reactive protein, IL-10, or IL-6 (all P > 0.05). Ambient BTEX exposure is associated with inflammation in mid-pregnancy in African-American women. Future studies examining if inflammation mediates associations between BTEX exposure and PTB are needed.

Macroautophagy/autophagy can play a cytoprotective role after photodynamic damage to malignant cells, depending on the site of subcellular damage initiated by reactive oxygen species. There is evidence for such protection when mitochondria are among the targets. Targeting lysosomes has been reported to be more effective for photokilling, perhaps because autophagy offers no cytoprotection. Photodynamic damage to both lysosomes and mitochondria can, however, markedly enhance the overall level of photokilling. Two mechanisms have been proposed to account for this result. Lysosomal photodamage leads to the release of calcium ions, resulting in the activation of the protease CAPN (calpain). CAPN then cleaves ATG5 to a fragment (tATG5) capable of interacting with mitochondria to enhance pro-apoptotic signals. It has also been proposed that targeting lysosomes for photodynamic damage can impair mitophagy, a process that could mitigate the pro-apoptotic effects of mitochondrial targeting. The level of lysosomal photodamage required for suppression of mitophagy is unclear. The "tATG5 route" involves the catalytic action of CAPN, activated by a degree of lysosomal photodamage barely detectible by a viability assay. ER photodamage can also initiate paraptosis, a death pathway functional even in cell types with impaired apoptosis and apparently unaffected by autophagy. Abbreviations: ALLN: N-acetyl-Leu-Leu-norleucinal (cell-permeable inhibitor of calpain); ATG: autophagy related; BPD: benzoporphyrin derivative (Visudyne); ER: endoplasmic reticulum; EtNBS: 5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride; MTT: a tetrazolium dye; NPe6: mono N-aspartyl chlorin e6; PDT: photodynamic therapy; ROS: reactive oxygen species.

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 < 0.001), but not PM2.5 (P = 0.376) or NO2 (P = 0.582), were statistically significantly associated with PTB. In adjusted models, for every 5-unit increase in PM10 there was a 1.21 times higher odds of PTB (95% CI 1.07, 1.38) and for every 5-unit increase in BTEX there was a 1.54 times higher odds of PTB (95% CI 1.25, 1.89). Consistent with previous studies, higher PM10 was associated with PTB. We also found novel evidence that higher airshed BTEX is associated with PTB. Future studies confirming these associations and examining direct measures of exposure are needed.

In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)- phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases. aDepartment of Pharmacology,Wayne State University School of Medicine, Detroit, MI, 48201, USA bCancer Biology Program, Wayne State University, Detroit, MI, 48201, USA cInstitute of Environmental Health Sciences, Eugene Applebaum Building, Wayne State University, Detroit, MI, 48201, USA Abbreviations BPD Benzoporphyrin derivative E64d 2S,3S-trans-(ethoxycarbonyloxirane-2-carbonyl)- L-leucine-(3-methylbutyl) amide (an inhibitor of calpain and cysteine protease activity) ER Endoplasmic reticulum LDxx PDT dose that reduces viability to xx% of controls LMP Lysosomal membrane permeabilization LSG LysoSensor Green mTHPC meso-Tetrahydroxyphenylporphine NPe6 N-Aspartyl chlorine e63-O-MeSM 3-O-Methyl sphingomyelin PCI Photochemical internalization PDT Photodynamic therapy PI Phosphatidyl inositol TDPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5- Triene

We previously reported that a low level of lysosomal photodamage potentiated the phototoxic effect of subsequent mitochondrial photodamage mediated by the benzoporphyrin derivative (BPD) in murine hepatoma 1c1c7 cells. This was attributed to release of Ca2+ from damaged lysosomes and a calpain‐mediated conversion of the autophagy‐related protein ATG5 to a pro‐apoptotic fragment. We now report a comparison of these results with those obtained with the human non‐small‐cell lung cancer A549 cell line. A549 cells contained lower levels of ATG5 and were less responsive than 1c1c7 cultures to the PDT combination. A rapid appearance of caspase 3/7 activation together with formation of condensed chromatin indicated initiation of apoptosis in both cell lines, but to a lesser extent in A549 cultures. Both cell lines became highly vacuolated within 16 h of combination PDT or an equivalent phototoxic dose from BPD alone. The vacuole periphery was labeled with a fluorescent probe for the endoplasmic reticulum (ER), and vacuole formation was prevented by presence of the protein synthesis inhibitor cycloheximide. These effects are characteristics of a caspase‐independent death mode termed paraptosis previously associated with ER stress. These studies suggest that paraptosis may be a more frequent outcome of PDT than has hitherto been realized.

Forty eight potential outliers in air pollution measurements taken simultaneously in Detroit, Michigan, USA and Windsor, Ontario, Canada in 2008 and 2009 were identified using four independent methods: box plots, variogram clouds, difference maps, and the Local Moran's I statistic. These methods were subsequently used in combination to reduce and select a final set of 13 outliers for nitrogen dioxide (NO2), volatile organic compounds (VOCs), total benzene, toluene, ethyl benzene, and xylene (BTEX), and particulate matter in two size fractions (PM2.5 and PM10). The selected outliers were excluded from the measurement datasets and used to revise air pollution models. In addition, a set of temporally-scaled air pollution models was generated using time series measurements from community air quality monitors, with and without the selected outliers. The influence of outlier exclusion on associations with asthma exacerbation rates aggregated at a postal zone scale in both cities was evaluated. Results demonstrate that the inclusion or exclusion of outliers influences the strength of observed associations between intraurban air quality and asthma exacerbation in both cities. The box plot, variogram cloud, and difference map methods largely determined the final list of outliers, due to the high degree of conformity among their results. The Moran's I approach was not useful for outlier identification in the datasets studied. Removing outliers changed the spatial distribution of modeled concentration values and derivative exposure estimates averaged over postal zones. Overall, associations between air pollution and acute asthma exacerbation rates were weaker with outliers removed, but improved with the addition of temporal information. Decreases in statistically significant associations between air pollution and asthma resulted, in part, from smaller pollutant concentration ranges used for linear regression. Nevertheless, the practice of identifying outliers through congruence among multiple methods strengthens confidence in the analysis of outlier presence and influence in environmental datasets.

Oncogenic Ras proteins are a driving force in a significant set of human cancers and wildtype, 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.

Oncogenic Ras proteins are a driving force in a significant set of human cancers and wildtype, 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. Keywords: H-Ras, K-Ras, lovastatin, N-Ras, prenylation inhibitors, statins, subcellular localization.

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.

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.

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 and activation of procaspases‐3 and ‐7. These inhibitors did not, however, suppress the proapoptotic 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 as pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdowns may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial proapoptotic processes. Our results suggest that low‐dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers.

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.

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. NO 2 , SO 2 , 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, NO 2 , VOCs, and PM 10 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 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 mu M) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with >= 100 mu M pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 mu M), 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 mu M pAA was cytostatic and induced a G(1) arrest that lasted similar to 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.

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.

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 LD50 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.