Reshma Bhowmick

Photoreceptor IFT Complexes Containing Chaperones, Guanylyl Cyclase 1 and Rhodopsin

Intraflagellar transport (IFT) provides a mechanism for the transport of cilium‐specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT–cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor‐specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT–cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg‐ATP enhances the co‐IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1‐green fluorescent protein (GFP) reduces cilium membrane targeting of GC1‐GFP without apparent effect on cilium elongation.

Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.

Cytoprotective signaling associated with nitric oxide upregulation in tumor cells subjected to photodynamic therapy-like oxidative stress

Photodynamic therapy (PDT) employs photoexcitation of a sensitizer to generate tumor-eradicating reactive oxygen species. We recently showed that irradiating breast cancer COH-BR1 cells after treating with 5-aminolevulinic acid (ALA, a pro-sensitizer) resulted in rapid upregulation of inducible nitric oxide (NO) synthase (iNOS). Apoptotic cell killing was strongly enhanced by an iNOS inhibitor (1400W), iNOS knockdown (kd), or a NO scavenger, suggesting that NO was acting cytoprotectively. Stress signaling associated with these effects was examined in this study. ALA/light-stressed COH-BR1 cells, and also breast adenocarcinoma MDA-MB-231 cells, mounted an iNOS/NO-dependent resistance to apoptosis that proved to be cGMP-independent. Immunocytochemistry and subcellular Western analysis of photostressed COH-BR1 cells revealed a cytosol-to-nucleus translocation of NF-κB which was negated by the NF-κB activation inhibitor Bay11. Bay11 also enhanced apoptosis and prevented iNOS induction, consistent with NF-κB involvement in the latter. JNK and p38 MAP kinase inhibitors suppressed apoptosis, implicating these kinases in death signaling. Post-irradiation extent and duration of JNK and p38 phosphorylation were dramatically elevated by 1400 W or iNOS-kd, suggesting that these activations were suppressed by NO. Regarding pro-survival stress signaling, rapid activation of Akt was unaffected by 1400 W, but prevented by Wortmannin, which also enhanced apoptosis. Thus, a link between upstream Akt activation and iNOS induction was apparent. Furthermore, p53 protein expression under photostress was elevated by iNOS-kd, whereas robust Survivin induction was abolished, consistent with p53 and Survivin being negatively and positively regulated by NO, respectively. Collectively, these findings enhance our understanding of cytoprotective signaling associated with photostress-induced NO and suggest iNOS inhibitor-based approaches for improving PDT efficacy.

Pro-survival and pro-growth effects of stress-induced nitric oxide in a prostate cancer photodynamic therapy model

We discovered recently that human breast cancer cells subjected to photodynamic therapy (PDT)-like oxidative stress localized in mitochondria rapidly upregulated nitric oxide synthase-2 (NOS2) and nitric oxide (NO), which increased resistance to apoptotic photokilling. In this study, we asked whether human prostate cancer PC-3 cells would exploit NOS2/NO similarly and, if so, how proliferation of surviving cells might be affected. Irradiation of photosensitized PC-3 cells resulted in a rapid (<1 h), robust (~12-fold), and prolonged (∼20 h) post-irradiation upregulation of NOS2. Caspase-3/7 activation and apoptosis were stimulated by NOS2 inhibitors and a NO scavenger, implying that induced NO was acting cytoprotectively. Cyclic GMP involvement was ruled out, whereas suppression of pro-apoptotic JNK and p38 MAPK activation was clearly implicated. Cells surviving photostress grew back ~2-times faster than controls. NOS2 inhibition prevented this and the large increase in cell cycle S-phase occupancy observed after irradiation. Thus, photostress upregulation of NOS/NO elicited both a pro-survival and pro-growth response, both of which could compromise clinical PDT efficacy unless suppressed, e.g. by pharmacological intervention with a NOS2 inhibitor.

SIGNALING EVENTS IN APOPTOTIC PHOTOKILLING OF 5-AMINOLEVULINIC ACID-TREATED TUMOR CELLS: INHIBITORY EFFECTS OF NITRIC OXIDE

Antitumor photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that can destroy tumor and tumor vasculature cells. NO produced by these cells could be procarcinogenic by inhibiting apoptosis and promoting angiogenesis and tumor growth. We recently showed that NO from a chemical donor or activated macrophages makes COH-BR1 breast tumor cells more resistant to photokilling sensitized by 5-aminolevulinic acid (ALA)-generated protoporphyrin IX (PpIX). Signaling events associated with this hyperresistance have now been examined. ALA-treated COH-BR1 cells containing mitochondria-localized PpIX died mainly by apoptosis after being irradiated. Underlying redox signaling associated with MAP kinase (ERK1/2, p38, JUN) phosphorylation-activation, and heme oxygenase-1 (HO-1) upregulation was studied using immunoprecipitation and Western blot methodology. ALA/light treatment resulted in activation of proapoptotic JNK and p38 alpha, and deactivation of prosurvival p38 beta and ERK1/2. Involvement of both JNK and p38 in apoptosis was established by using a specific inhibitor for each. Spermine NONOate-derived NO, introduced immediately before irradiation, provided substantial protection against apoptosis. This was accompanied by greater HO-1 induction and a strong inhibition of each MAP kinase effect seen in the absence of NO. Downstream of JNK and p38 alpha activation, a marked upregulation/activation of proapoptotic Bax and Bid was observed along with down-regulation of antiapoptotic Bcl-xL, each response being reversed by NO. These findings provide new insights into signaling activity associated with the intrinsic apoptotic pathway in ALA-PDT and how this activity can be modulated by NO.

Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge.

Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH‐BR1 breast cancer cells sensitized in mitochondria with 5‐aminolevulinic acid (ALA)‐generated protoporphyrin IX as a model for ALA‐based PDT, we found that caspase‐9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT‐PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W‐inhibitable increase in intracellular NO, as detected with the NO probe, DAF‐2‐DA. Short hairpin RNA‐based iNOS knockdown in COH‐BR1 cells dramatically reduced NO production under photostress while enhancing caspase‐9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT‐treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.

Impairments in age-dependent ubiquitin proteostasis and structural integrity of selective neurons by uncoupling Ran GTPase from the Ran-binding domain 3 of Ranbp2 and identification of novel mitochondrial isoforms of ubiquitin-conjugating enzyme E2I (ubc9) and Ranbp2

ABSTRACT The Ran-binding protein 2 (Ranbp2/Nup358) is a cytoplasmic and peripheral nucleoporin comprised of 4 Ran-GTP-binding domains (RBDs) that are interspersed among diverse structural domains with multifunctional activities. Our prior studies found that the RBD2 and RBD3 of Ranbp2 control mitochondrial motility independently of Ran-GTP-binding in cultured cells, whereas loss of Ran-GTP-binding to RBD2 and RBD3 are essential to support cone photoreceptor development and the survival of mature retinal pigment epithelium (RPE) in mice. Here, we uncover that loss of Ran-GTP-binding to RBD3 alone promotes the robust age-dependent increase of ubiquitylated substrates and S1 subunit (Pmsd1) of the 19S cap of the proteasome in the retina and RPE and that such loss in RBD3 also compromises the structural integrity of the outer segment compartment of cone photoreceptors only and without affecting the viability of these neurons. We also found that the E2-ligase and partner of Ranbp2, ubc9, is localized prominently in the mitochondrial-rich ellipsoid compartment of photoreceptors, where Ranbp2 is also known to localize with and modulate the activity of mitochondrial proteins. However, the natures of Ranbp2 and ubc9 isoforms to the mitochondria are heretofore elusive. Subcellular fractionation, co-immunolocalization and immunoaffinity purification of Ranbp2 complexes show that novel isoforms of Ranbp2 and ubc9 with molecular masses distinct from the large Ranbp2 and unmodified ubc9 isoforms localize specifically to the mitochondrial fraction or associate with mitochondrial components, whereas unmodified and SUMOylated Ran GTPase are excluded from the mitochondrial fraction. Further, liposome-mediated intracellular delivery of an antibody against a domain shared by the mitochondrial and nuclear pore isoforms of Ranbp2 causes the profound fragmentation of mitochondria and their delocalization from Ranbp2 and without affecting Ranbp2 localization at the nuclear pores. Collectively, the data support that Ran GTPase-dependent and independent and moonlighting roles of Ranbp2 or domains thereof and ubc9 control selectively age-dependent, neural-type and mitochondrial functions.

Abstract A45: Signaling Events Associated with Cytoprotective Induction of Nitric Oxide Synthase in a Photodynamic Therapy Model

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and photosensitizer-exciting light to produce reactive oxygen species that lead to tumor eradication. Nitric oxide (NO) produced by tumor or tumor vasculature cells could be pro-carcinogenic by inhibiting apoptosis and/or promoting angiogenesis and tumor growth. We recently showed that photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX in mitochondria of COH-BR1 breast tumor cells strongly upregulated inducible nitric oxide synthase (iNOS) and steady state NO level in these cells. Including the iNOS inhibitor 1400W during photochallenge dramatically enhanced caspase-9 activation and Annexin-assessed apoptotic cell killing while reducing NO production assessed by the intracellular NO probe DAF-2DA. Short hairpin RNA (ShRNA)-based iNOS knockdown (kd) produced similar results, confirming iNOS involvement. An apoptosis-promoting effect of iNOS inhibition was also observed when breast MDA-MB231 and prostate PC-3 cancer cells were subjected to ALA/light stress. We have now examined the signaling events associated with iNOS-mediated hyperresistance of COH-BR1 cells using a combination of immunocytochemistry, western blotting, immunoprecipitaion, and iNOS-kd methods. A soluble guanylyl cyclase inhibitor failed to stimulate ALA/light-provoked apoptosis, ruling out cGMP involvement in stress resistance. ALA/light activated PI3-kinase-dependent signaling via phosphorylation-inactivation of the tumor suppressor PTEN, leading to phosphorylation-activation of pro-survival Akt. Inhibition of PI3K by Wortmannin prevented ALA/light-induced Akt activation as well as iNOS upregulation while enhancing apoptotic photokilling. Moreover, photostress activation of PI3K/Akt was accompanied by a cytosol-to-nucleus translocation of the iNOS transcription factor NF-κB, and an inhibitor of NF-kB activation prevented iNOS induction while stimulating apoptosis. Furthermore, iNOS-kd as well as 1400W treatment resulted in intensified and more prolonged activation of MAP kinases JNK and p38α by ALA/light stress. In addition, photostressed iNOS-kd cells exhibited p53 upregulation and Survivin inactivation/downregulation. The following general mechanism of NO-based cytoprotection is suggested from these and related findings: Photostress activation of PI3K/Akt suppresses pro-apoptotic MAP kinases and p53 while upregulating/activating Survivin via NF-κB-mediated induction of iNOS/NO. This study provides important new insights into photostress-elicited pro-survival signaling that could reduce PDT effectiveness, and suggests iNOS inhibitor-based interventions for counteracting this. (Supported by NIH Grant CA70823 and a grant from the MCW Cancer Center.)

Abstract 132: Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that kill tumor and tumor vasculature cells. Nitric oxide (NO) produced by these cells could be pro-carcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. Our previous studies showed that NO from a chemical donor made COH-BR1 breast tumor cells hyperresistant to apoptotic photokilling induced by photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX localized in mitochondria. Hyperresistance was associated with inhibition of proapoptotic JNK and p38α MAP kinase phosphorylation-activation. The purpose of this study was to determine whether tumor cells upregulate NOS-generated NO as a cytoprotective measure during PDT.COH-BR1 cells sensitized in mitochondria with ALA-derived protoporphyrin IX died apoptotically following irradiation as measured by Hoechst staining. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, while other NOS isoforms were unaffected. Exposing cells to the iNOS inhibitor, 1400W during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2-3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. Using Hoechst and TUNEL staining, we found that short hairpin RNA (shRNA)-induced knockdown of iNOS enhanced the apoptotic kill of ALA/light treated COH-BR1 cells. ALA/light-treated COH-BR1cells exhibited a transient post-irradiation activation of JNK and p38α as measured by Western blot analysis. Consistently, both effects were intensified and prolonged by 1400W. The survival MAP kinase ERK1/2 was deactivated more rapidly when 1400W was present during a PpIX/light challenge. Similar effects on MAP kinase activation/deactivation were observed for iNOS knockdown cells under photostress cinfirming iNOS9s protective role. As demonstrated for non-photodynamic stress systems, NO could have interfered with apoptosis by inactivating participating MAP kinases and/or caspases. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy, and suggest NOS-based pharmacologic interventions for preventing this. (Supported by NIH Grant CA70823) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 132.