Thomas H. March

Histone Deacetylase 2 Is Phosphorylated, Ubiquitinated, and Degraded by Cigarette Smoke

Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Respiratory Tract

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the respiratory tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the inter-net (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for respiratory tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Pulmonary Prostacyclin Synthase Overexpression Chemoprevents Tobacco Smoke Lung Carcinogenesis in Mice

Increased pulmonary production of prostaglandin I2 (prostacyclin) by lung-specific overexpression of prostacyclin synthase decreases lung tumor incidence and multiplicity in chemically induced murine lung cancer models. We hypothesized that pulmonary prostacyclin synthase overexpression would prevent lung carcinogenesis in tobacco-smoke exposed mice. Murine exposure to tobacco smoke is an established model of inducing pulmonary adenocarcinomas and allows for the testing of potential chemopreventive strategies. Transgenic FVB/N mice with lung-specific prostacyclin synthase overexpression were exposed to mainstream cigarette smoke for 22 weeks and then held unexposed for an additional 20 weeks. All of the exposed animals developed bronchiolitis analogous to the respiratory bronchiolitis seen in human smokers. The transgenic mice, when compared with smoke-exposed transgene negative littermates, had significant decreases in tumor incidence and multiplicity. Significantly fewer transgenics (6 of 15; 40%) developed tumors compared with the tumor incidence in wild-type littermates (16 of 19; 84%; Fisher’s exact test, P = 0.012). Tumor multiplicity was also significantly decreased in the transgenic animals (tg+ = 0.4 ± 0.5 versus wild-type = 1.2 ± 0.86 tumors/mouse; P < 0.001). Targeted prostaglandin levels at the time of sacrifice revealed significantly elevated prostaglandin I2 levels in the transgenic animals, coupled with significantly decreased prostaglandin E2 levels. Gene expression analysis of isolated type II pneumocytes suggests potential explanations for the observed chemoprevention, with Western blot analysis confirming decreased expression of cytochrome p450 2e1. These studies extend our previous studies and demonstrate that manipulation of prostaglandin production distal to cyclooxygenase significantly reduces lung carcinogenesis in a tobacco smoke exposure model, and gene expression studies show critical alterations in antioxidation, immune response, and cytokine pathways.

EFFECTS OF CIGARETTE SMOKE EXPOSURE AND CESSATION ON INFLAMMATORY CELLS AND MATRIX METALLOPROTEINASE ACTIVITY IN MICE

B6C3F1 female mice were exposed to cigarette smoke (CS) (250 mg/m3 total particulate material) or filtered air (FA), 6 hours/day, 5 days/week, for 6, 7, or 10 weeks, or to CS for 6 weeks, then FA for 1 or 4 additional weeks. Exposure to CS increased macrophages, neutrophils, lymphocytes, and matrix metalloproteinase (MMP)-2 and MMP-9 content in bronchoalveolar lavage fluid. Partial recovery of most lavage parameters (except lymphocytes) was observed 1 week after cessation of CS exposure with further reductions after 4 weeks, but interstitial inflammation persisted longer. These results support a role for MMPs in CS-induced emphysema and indicate that smoking cessation allows restoration toward normal homeostasis.

Combination Therapy with Vidaza and Entinostat Suppresses Tumor Growth and Reprograms the Epigenome in an Orthotopic Lung Cancer Model

Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of proapoptotic genes and the cyclin-dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the reexpression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings show the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs.

Effects of Strain and Treatment with Inhaled All-Trans-Retinoic Acid on Cigarette Smoke-Induced Pulmonary Emphysema in Mice

Models of emphysema produced by exposing animals to cigarette smoke (CS) have potential for use in testing treatments of this disease. To better characterize development of emphysema in an animal model, male and female mice of the B6C3F1 and A/J strains were exposed to CS at 250 mg total particulate material (TPM)/m3 for 15 weeks. Emphysema was evident in both strains of mice to differing degrees of severity. The CS-induced increase in the mean linear intercept (normalized to BW) of A/J mice was 51% greater than the control value, while CS-exposed B6C3F1 had an increase of 38% in this morphometric measurement of alveolar air space enlargement. In separate experiments, female B6C3F1 mice and male A/J mice were exposed to CS for 32 weeks and 15 weeks, respectively, and were then used to test the efficacy of all trans-retinoic acid (ATRA) treatments to ameliorate emphysema lesions. Following CS exposure, the B6C3F1 mice were treated once daily for 14 days in a 3-week period by nose-only inhalation exposure to aerosols of 180 or 1,800 mg-minutes ATRA/m3. The A/J mice were treated once daily, 4 days/week, for three weeks by either intraperitoneal injection of ATRA (0.5 or 2.5 mg/kg) or inhalation exposure to ATRA (3,600 or 18,000 mg-minutes/m3). Neither the injections nor inhalation exposures of ATRA in either strain of mouse caused reversal of the emphysema. In summary, CS-induced emphysema was more severe in A/J mice than in B6C3F1 mice. Treatment with ATRA did not reverse emphysema in either strain of CS-exposed mice.

Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assessed possible adverse health effects associated with inhalation exposure to brevetoxin 3, one of the major brevetoxins produced by K. brevis and present in aerosols collected along beaches affected by red tide. Male F344 rats were exposed to brevetoxin 3 at 0, 37, and 237 μg/m3 by nose-only inhalation 2 hr/day, 5 days/week for up to 22 exposure days. Estimated deposited brevetoxin 3 doses were 0.9 and 5.8 μg/kg/day for the low-and high-dose groups, respectively. Body weights of the high-dose group were significantly below control values. There were no clinical signs of toxicity. Terminal body weights of both low- and high-dose-group rats were significantly below control values. Minimal alveolar macrophage hyperplasia was observed in three of six and six of six of the low- and high-dose groups, respectively. No histopathologic lesions were observed in the nose, brain, liver, or bone marrow of any group. Reticulocyte numbers in whole blood were significantly increased in the high-dose group, and mean corpuscular volume showed a significant decreasing trend with increasing exposure concentration. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by significant reduction in splenic plaque-forming cells in both low- and high-dose-group rats compared with controls. Results indicate that the immune system is the primary target for toxicity in rats after repeated inhalation exposure to relatively high concentrations of brevetoxins.

INHALATION ADMINISTRATION OF ALL-TRANS-RETINOIC ACID FOR TREATMENT OF ELASTASE-INDUCED PULMONARY EMPHYSEMA IN FISCHER 344 RATS

A past study demonstrated that all-trans-retinoic acid (ATRA) treatment by intraperitoneal injection in a rat model of elastase-induced emphysema caused tissue regeneration as evidenced by a decrease in alveolar size and lung volume and an increase in alveolar number. We postulated that treatment with this retinoid by nose-only inhalation exposure would be a more efficient means of targeting damaged lung tissue. Emphysema was induced in male Fischer 344 rats by intratracheal instillation of pancreatic elastase (0.5 IU/g body weight). Four weeks after elastase instillation, animals were treated once daily, 4 days/week, for 3 weeks by exposing them nose-only to aerosolized ATRA (target concentration-time of 3000 or 15,000 mg-min/m3) or by injecting them intraperitoneally with ATRA in cottonseed oil (0.5 or 2.5 mg/kg). Based on estimates of particle deposition in the respiratory tract, inhalation doses were chosen to be consistent with injected doses. Lungs were fixed by inflation with formalin (constant pressure for 6 hours followed by >48 hours of immersion) and were embedded in paraffin. Sections were evaluated by histopathology and stereology. Inhalation exposure to ATRA at both aerosol concentrations caused significant elevations of ATRA in the lung, whereas only the high-dose injection treatment was associated with an elevation of lung ATRA. The mean ATRA concentration from lungs of rats in the high-dose inhalation exposure groups as measured by liquid chromatography–mass spectrometry was approximately 12-fold greater than that of high-dose injection-treated rats. Elastase instillation caused increased lung volumes, irregular alveolar air space enlargement, and fragmentation and attenuation of alveolar septa. Neither inhaled nor injected ATRA reduced the enlarged lung volumes associated with this emphysema model. Stereology demonstrated that alveolar air space enlargement in ATRA-treated rats was similar to that in sham-treated emphysematous animals. Thus, while inhalation treatment caused greater levels of the drug in lung tissue in comparison to that of injection-treated animals, treatment with ATRA by either route of administration did not cause a reversal of lung tissue damage in this model of elastase-induced emphysema.

SGI-110 and entinostat therapy reduces lung tumor burden and reprograms the epigenome

The DNA methyltransferase (DNMT) inhibitor vidaza (5‐Azacytidine) in combination with the histone deacetylase inhibitor entinostat has shown promise in treating lung cancer and this has been replicated in our orthotopic lung cancer model. However, the effectiveness of DNMT inhibitors against solid tumors is likely impacted by their limited stability and rapid inactivation by cytidine deaminase (CDA) in the liver. These studies were initiated to test the efficacy of SGI‐110, a dinucleotide containing decitabine that is resistant to deamination by CDA, as a single agent and in combination with entinostat. Evaluation of in vivo plasma concentrations and pharmacokinetic properties of SGI‐110 showed rapid conversion to decitabine and a plasma half‐life of 4 hr. SGI‐110 alone or in combination with entinostat reduced tumor burden of a K‐ras/p53 mutant lung adenocarcinoma cell line (Calu6) engrafted orthotopically in nude rats by 35% and 56%, respectively. SGI‐110 caused widespread demethylation of more than 300 gene promoters and microarray analysis revealed expression changes for 212 and 592 genes with SGI‐110 alone or in combination with entinostat. Epigenetic therapy also induced demethylation and expression of cancer testis antigen genes that could sensitize tumor cells to subsequent immunotherapy. In the orthotopically growing tumors, highly significant gene expression changes were seen in key cancer regulatory pathways including induction of p21 and the apoptotic gene BIK. Moreover, SGI‐110 in combination with entinostat caused widespread epigenetic reprogramming of EZH2‐target genes. These preclinical in vivo findings demonstrate the clinical potential of SGI‐110 for reducing lung tumor burden through reprogramming the epigenome.

Rosiglitazone prevents the progression of preinvasive lung cancer in a murine model

There is a critical need to identify efficacious chemopreventive agents for lung cancer that can be taken chronically with no side effects and whose mechanisms of action do not involve genotoxicity that could drive, rather than impede, cancer progression. We evaluated the ability of a chemopreventive cocktail that included selenium (antioxidant), rosiglitazone (peroxisome proliferator-activated receptor gamma agonist), sodium phenylbutyrate or valproic acid (histone deacetylase inhibitors) and hydralazine (cytosine-demethylating agent) to prevent the progression of lung cancer in A/J mice treated with NNK. Agents were administered alone or in various combinations. Effects of the chemopreventive agents were quantified based on the proportion of hyperplasias and adenomas within the mouse lung. Significant effects on tumor progression were seen in all treatment groups that included rosiglitazone as reflected by a 47-57% increase in number of hyperplasias and a 10-30% decrease in adenomas. Cell proliferation was also reduced in these treatment groups by approximately 40%. Interestingly, while treatment with rosiglitazone alone did not significantly affect lesion size, striking effects were seen in the combination therapy group that included sodium phenylbutyrate, with the volume of hyperplasias and adenomas decreasing by 40 and 77%, respectively. These studies demonstrate for the first time that chronic in vivo administration of rosiglitazone, used in the management of diabetes mellitus, can significantly block the progression of premalignant lung cancer in the A/J mouse model.