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Supplementary Tables 1-2, Figures 1-5 from Cancer Causes Cardiac Atrophy and Autophagy in a Sexually Dimorphic Manner

Approximately one-third of cancer deaths are caused by cachexia, a severe form of skeletal muscle and adipose tissue wasting that affects men more than women. The heart also undergoes atrophy in cancer patients, but the mechanisms and the basis for apparent sex differences are unclear. In a mouse colon-adenocarcinoma model, cancer causes a loss of cardiac mass due to a decrease in cardiac myocyte size that is associated with reduced levels of all sarcomeric proteins. Unlike skeletal muscle cachexia, atrophic hearts do not upregulate the ubiquitin-proteasome system or its activity but increase autophagy. Thus, cancer causes cardiac atrophy by a mechanism distinct from that in skeletal muscle. Male tumor-bearing mice have a more severe phenotype than females, including greater cardiac mass loss and mortality, a more robust pro-inflammatory response to the tumor, and greater cardiac autophagy. In females, estrogen protects against cancer-induced cardiac atrophy and body weight loss by signaling through its receptor. Sex differences in cardiac atrophy need to be considered during the treatment of patients suffering from chemotherapy-induced cardiomyopathy to prevent exacerbation of cardiac dysfunction. Cancer Res; 71(5); 1710–20. ©2010 AACR.

Purpose Cervical cancer represents the fourth most frequent malignancy in the world among women, and mortality has remained stable for the past 4 decades. Intravenous cisplatin with concurrent radiation therapy is the standard-of-care for patients with local and regional cervical cancer. However, cisplatin induces serious dose-limiting systemic toxicities and recurrence frequently occurs. In this study, we aimed to develop an intracervical drug delivery system that allows cisplatin release directly into the tumor and minimize systemic side effects. Methods and Materials Twenty patient biopsies and 5 cell lines treated with cisplatin were analyzed for platinum content using inductively coupled plasma mass spectrometry. Polymeric implants loaded with cisplatin were developed and evaluated for degradation and drug release. The effect of local or systemic cisplatin delivery on drug biodistribution as well as tumor burden were evaluated in vivo, in combination with radiation therapy. Results Platinum levels in patient biopsies were 6-fold lower than the levels needed for efficacy and radiosensitization in vitro. Cisplatin local delivery implant remarkably improved drug specificity to the tumor and significantly decreased accumulation in the blood, kidney, and other distant normal organs, compared with traditional systemic delivery. The localized treatment further resulted in complete inhibition of tumor growth. Conclusions The current standard-of-care systemic administration of cisplatin provides a subtherapeutic dose. We developed a polymeric drug delivery system that delivered high doses of cisplatin directly into the cervical tumor, while lowering drug accumulation and consequent side effects in normal tissues. Moving forward, these data will be used as the basis of a future first-in-human clinical trial to test the efficacy of localized cisplatin as adjuvant or neoadjuvant chemotherapy in local and regional cervical cancer.

Chromosome missegregation over the course of multiple cell divisions, termed chromosomal instability (CIN), is a hallmark of cancer. Multiple causes of CIN have been identified, including defects in the mitotic checkpoint, altered kinetochore-microtubule dynamics, centrosome amplification, and ionizing radiation. Here we review the types, mechanisms, and cellular implications of CIN. We discuss the evidence that CIN can promote tumors, suppress them, or do neither, depending on the rates of chromosome missegregration and the cellular context. Very high rates of chromosome missegregation lead to cell death due to loss of essential chromosomes; thus elevating CIN above a tolerable threshold provides a mechanistic opportunity to promote cancer cell death. Lethal rates of CIN can be achieved by a single insult or through a combination of insults. Because ionizing radiation induces CIN, additional therapies that increase CIN may serve as useful modulators of radiation sensitivity. Ultimately, quantifying the intrinsic CIN in a tumor and modulating this level pharmacologically as well as with radiation may allow for a more rational, personalized radiation therapy prescription, thereby decreasing side effects and increasing local control.

Human papillomavirus (HPV) is a ubiquitous DNA virus that infects squamous epithelia. Though HPV only encodes 8 genes, it is capable of causing cellular transformation and ultimately cancer in host cells. In this article we review the classification of HPV viruses, their genetic structure and life cycle, viral gene biology, and provide an overview of the role of HPV in cancer. We explain how the viral life cycle can lead to integration of viral DNA into the host genome leading to increased cell cycle progression, decreased apoptosis, altered DNA repair, and chromosomal instability. We describe the multifaceted roles of the canonical oncogenes E6 and E7 in promoting tumorigenesis and the important role of other viral genes in regulating cancer development. We also review how the virus actively suppresses innate and adaptive immunity to evade immune detection and promote a pro-tumorigenic microenvironment. The biology presented here will serve as a foundation to the other chapters in this edition and we hope it will incite enthusiasm for continued research on this fascinating virus that causes significant morbidity and mortality worldwide.

More than one-third of patients with locally advanced cervical cancer do not respond to chemoradiation therapy (CRT). We aimed to characterize the transcriptional landscape of paired human cervical tumors before and during CRT in order to gain insight into the evolution of treatment response and to elucidate mechanisms of treatment resistance. We prospectively collected cervical tumor biopsies from 115 patients both before and 3 weeks into CRT. RNA-sequencing, Gene Set Enrichment Analysis and HPV gene expression were performed on 20 paired samples that had adequate neoplastic tissue mid-treatment. Tumors from patients with no evidence of disease (NED) at last follow-up had enrichment in pathways related to the immune response both pretreatment and mid-treatment, while tumors from patients dead of disease (DOD) demonstrated enrichment in biosynthetic and mitotic pathways but not in immune-related pathways. Patients DOD had decreased expression of T-cell and cytolytic genes and increased expression of PD-L2 mid-treatment compared to patients NED. Histological and immunohistochemical analysis revealed a decrease in tumor-associated lymphocytes (TAL) during CRT in all patients but tumors from patients DOD had a significantly more pronounced decrease in TALs and CD8+ cells mid-treatment, which was validated in a larger mid-treatment cohort. Finally, patients DOD retained more HPV E6/E7 gene expression during CRT and this was associated with increased expression of genes driving mitosis, which was corroborated in vitro. Our results suggest that decreased local immune response and retained HPV gene expression may be acting together to promote treatment resistance during CRT in patients with cervical cancer.

Background: Cetuximab (CTX) is a monoclonal antibody commonly used in the treatment of head and neck squamous cell carcinomas (HNSCCs) that acts by targeting the epidermal growth factor receptor (EGFR). Addition of CTX to standard multi-agent chemotherapy only slightly improves the response rate from 20% to 36%, highlighting the urgent need for improved treatment. Autophagy is a naturally occurring mechanism activated within cells to protect them from harsh conditions or stress but can also provide cancerous cells with protection against further treatment attempts. Here, we examine the efficacy of combining CTX with the autophagy inhibitor, SAR405, as well as investigating the specific mechanism of CTX-induced autophagy. Methods: To measure autophagy, we utilized the Promega LC3 reporter assay, Western blotting for LC3, immunofluorescence for LC3, and an acridine orange assay. We ran our assays using the HNSCC cell lines A253 (HPV-), UM-SCC1 (HPV+), and its CTX-resistant derivative, UM-SCC1-C5 (HPV+). Clonogenic assays were run to measure colony formation, cell counting kit 8 (CCK8) assays to determine cell survival, and flow cytometry to investigate cell cycle status. Results: In this study, we first were able to demonstrate that CTX can activate pro-survival autophagy. CTX-treated cells had higher LC3 flux and more LC3 puncta than control cells as assessed by western blot and immunofluorescence staining, respectively. CTX-resistant cells demonstrated higher basal LC3 flux compared to the CTX-sensitive parental cells. We also discovered that the use of the autophagy inhibitor, SAR405, could help improve tumor response when combined with CTX. The addition of SAR405 to CTX treatment led to a decrease in colony formation and a decrease in cell survival, overcoming resistance to CTX. Interestingly, we discovered that SAR405 alone led to cell cycle arrest in the G1 phase but did not lead to a statistically significant decrease in cell survival compared to the control, showing that SAR405 is able to arrest cell growth but is not cytotoxic. Finally, knockdown of EGFR or LAPTM4B prevented CTX-induced activation of autophagy. Conclusion: Through our work, we have been able to demonstrate that CTX is able to activate autophagy, and that this initiation of autophagy plays a cytoprotective role in HNSCC cells. The addition of the autophagy inhibitor SAR405 improved response to CTX treatment. The promising results obtained thus far open the door to future studies investigating the efficacy of combining CTX with more specific autophagy inhibitors. Citation Format: Samantha T. Bradley, Yong-Syu Lee, Justin H. Skiba, Jaimee Eckers, Adam D. Swick, Rong Hu, Kwang P. Nickel, Zafer Gurel, Pippa F. Cosper, Randall J. Kimple. The effects of autophagy inhibition on HNSCC sensitivity to CTX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3003.

Patient-derived model systems are important tools for studying novel anti-cancer therapies. Patient-derived xenografts (PDXs) have gained favor over the last 10 years as newer mouse strains have improved the success rate of establishing PDXs from patient biopsies. PDXs can be engrafted from head and neck cancer (HNC) samples across a wide range of cancer stages, retain the genetic features of their human source, and can be treated with both chemotherapy and radiation, allowing for clinically relevant studies. Not only do PDXs allow for the study of patient tissues in an in vivo model, they can also provide a renewable source of cancer cells for organoid cultures. Herein, we review the uses of HNC patient-derived models for radiation research, including approaches to establishing both orthotopic and heterotopic PDXs, approaches and potential pitfalls to delivering chemotherapy and radiation to these animal models, biological advantages and limitations, and alternatives to animal studies that still use patient-derived tissues.

Clinical outcomes and general treatment paradigms for patients with oral cavity, laryngeal, hypopharyngeal, and human papillomavirus (HPV) negative oropharyngeal squamous cell carcinoma (OPSCC) have remained relatively stable over the past several decades. Since the early 2000s it has become clear that patients with HPV + OPSCC have significantly improved outcomes compared to those with HPV − disease. This has led to considerable efforts being focused on defining the best approach to maximize the therapeutic ratio in patients with HPV + OPSCC in order to minimize the toxicities associated with treatment. Simultaneously, for patients with HPV − disease, attempts to intensify treatment have been taken in order to improve outcomes in patients with a significantly worse prognosis. In this chapter we review the role of radiation therapy in the treatment of head and neck cancer by focusing on the basics of radiobiology juxtaposed with the intricacies of modern radiotherapy, surgical techniques, systemic agents, and the on-going efforts aimed at combining their interactions to improve clinical outcomes while reducing treatment-related toxicities.

Squamous cell carcinoma antigen (SERPINB3) is a widely used biomarker for epithelial cancers including uterine cervix, head and neck, and esophagus. We have previously shown that SERPINB3 contributes to radiation resistance, mainly due to its predominant function as a protease inhibitor that interfered with lysosomal cell death. In addition, elevated SERPINB3 is also observed in various inflammatory conditions related to increased cytokine expression such as, atopic dermatitis, psoriasis, asthma and chronic obstructive pulmonary disease, indicative of potential immunoregulatory function of SERPINB3. We hypothesized that elevated SERPINB3 may also contribute to altered tumor immunity, leading to reduced sensitivity and therapeutic effects of radiation treatment. Thus, we sought evidence of SERPINB3 expression in relation to immune regulation using RNA sequencing analysis of cervical tumor biopsies from 66 patients, and identified a positive correlation of SERPINB3 with chemokines CXCL1, CXCL8, S100A8, and S100A9. These chemokines are known to induce myeloid cell accumulation in the tumor microenvironment, and the presence of myeloid cells have been shown to reduce the efficacy of radiotherapy in cervical cancer. Indeed we found enrichment of myeloid cell subsets on immune cell prediction analysis with xCell in SERPINB3-high tumors. We then examined expression and secretion of myeloid-attractant chemokines via quantitative PCR and ELISA in SERPINB3-overexpressing cervical cancer cells, and confirmed the upregulation of CXCL1/8 and S100A8/A9. Supernatants collected from SERPINB3-overexpressing cells contained chemoattractant to myeloid cells as evidenced by transwell assay using human peripheral blood mononuclear cells from patients with cervical cancer. In the mouse models, tumors formed by SERPINB3-expressing cells induced an immunosuppressive microenvironment with the increased infiltration of M2 macrophages and MDSCs in tumors and spleens. Furthermore, we observed the elevated phosphorylation of STAT1/3/5 transcription factors, known to regulate inflammatory chemokine expression, in SERPINB3-high cells. In our patient cohort, RNA-seq analysis of biopsies collected from 20 patients both before and 3 weeks into chemoradiation therapy (CRT) were analyzed for changes of SERPINB3 in comparison to chemokine expression. We found that patients with decreased SERPINB3 expression in the mid-CRT biopsy (n=7) showed downregulated S100A8/A9 levels in tumors while patients with increased SERPINB3 (n=13) had upregulated S100A8/A9 expression. Our findings suggest that SERPINB3 promotes chemokine secretion involved in myeloid cell-mediated immune suppression which may contribute to negative impacts on radiation treatment in patients with cervical cancer. Citation Format: Liyun Chen, Songyan Wang, Victoria Shi, Fiona Ruiz, Kay Jayachandran, Jin Zhang, Pippa Cosper, Perry Grigsby, Julie Schwarz, Stephanie Markovina. SERPINB3 promotes myeloid cell chemotaxis and associated chemokine production in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2680.

Background and Objectives. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas with poor overall survival. Response to chemotherapy has been debated for these tumors. Methods. We performed a retrospective analysis of the patients at our institution with a biopsy-proven diagnosis of MPNST that underwent neoadjuvant chemotherapy prior to surgery. Results. We retrospectively identified five patients who received neoadjuvant chemotherapy with epirubicin and ifosfamide that demonstrated a 30% reduction in tumor growth and a 60% response rate by RECIST criteria. Additionally, a metabolic response was observed in all three patients who received serial PET scans during neoadjuvant treatment. The clinical benefit rate, which includes stable disease, was 100%. Conclusions. Our data suggest that MPNSTs do respond to epirubicin and ifosfamide based chemotherapy and prospective studies are warranted to further define the clinical benefit.

Purpose We report the toxicity of patients treated with strut-adjusted volume implant (SAVI) for accelerated partial breast irradiation treated at our institution. Methods and Materials Patients treated from January 2013 to July 2015 with SAVI planned for 10 b.i.d. fractions for a total dose of 34 Gy were included. Acute and late toxicities were prospectively collected on patients in followup and graded by the Common Terminology Criteria for Adverse Events, version 4.0. Results A total of 132 patients were included, with 1 patient having synchronous breast cancer treated in each breast. Median followup was 20.0 months (range, 2.7–37.4 months). The median age at diagnosis was 61 years (range, 41–83 years). Forty-two lesions (32%) were in situ, 88 lesions (66%) were Stage 1, and 3 (2%) lesions were Stage 2. The median planning target volume was 58.2 cc (range, 24.2–109.9 cc), median V150 was 26.3 cc (range, 11.5–47.5 cc), and median V200 was 13.0 cc (range, 6.3–26.1 cc). On a pain scale of 0–10 (10 = worst pain), pain was worst on Day 2 of treatment, with an average score of 0.46. There was one acute skin infection; there were three late skin infections, two of which was Grade 3. Other late toxicities were Grade 1 or 2: hyperpigmentation (44%), telangiectasia (0.8%), seroma (9%), fat necrosis (5%), and fibrosis (12%). Crude local recurrence rate was 4%. Conclusion SAVI is a safe treatment option for patients who are candidates for accelerated partial breast irradiation. Local control seems to be excellent, but longer followup is needed.

Approximately one-third of cancer deaths are caused by cachexia, a severe form of skeletal muscle and adipose tissue wasting that affects men more than women. The heart also undergoes atrophy in cancer patients, but the mechanisms and the basis for apparent sex differences are unclear. In a mouse colon-adenocarcinoma model, cancer causes a loss of cardiac mass due to a decrease in cardiac myocyte size that is associated with reduced levels of all sarcomeric proteins. Unlike skeletal muscle cachexia, atrophic hearts do not upregulate the ubiquitin-proteasome system or its activity but increase autophagy. Thus, cancer causes cardiac atrophy by a mechanism distinct from that in skeletal muscle. Male tumor-bearing mice have a more severe phenotype than females, including greater cardiac mass loss and mortality, a more robust pro-inflammatory response to the tumor, and greater cardiac autophagy. In females, estrogen protects against cancer-induced cardiac atrophy and body weight loss by signaling through its receptor. Sex differences in cardiac atrophy need to be considered during the treatment of patients suffering from chemotherapy-induced cardiomyopathy to prevent exacerbation of cardiac dysfunction. Cancer Res; 71(5); 1710–20. ©2010 AACR.

Objective To determine whether it would be preferable to prescribe oral or transdermal estrogen to symptomatic postmenopausal women with metabolic syndrome (MBS). Design Prospective, randomized study. Setting Academic medical center. Patient(s) Fifty obese postmenopausal women with MBS. Intervention(s) Women were randomized to receive either oral E 2 (oE 2 , 1 mg/d) or transdermal E 2 (tE 2 , 0.05 mg/d) for 3 months. Fasting blood was obtained before and after treatment for glucose, insulin, lipid profiles, the adipocytokines (adiponectin, leptin, and resistin), and a gastric peptide (ghrelin). In addition, a 75-g 2-hour oral glucose-tolerance and intravenous insulin-tolerance tests were performed before and after E 2 . Main Outcome Measure(s) Changes in parameters of insulin resistance (IR), lipid profiles, and adipocytokine levels. Result(s) Mean serum concentrations of E 2 in women using oE 2 and tE 2 were 39.1 ± 5.6 and 49.2 ± 28.6 pg/mL, respectively. After oE 2 , there was a statistically significant worsening of IR markers, including an increase in baseline insulin (15.28 ± 1.27 to 22.02 ± 2.40 μU/mL), a reduction in quantitative insulin-sensitivity check index (0.3177 ± 0.0043 to 0.2977 ± 0.0057), and an increase in homeostasis model assessment (3.96 ± 0.38 to 8.59 ± 2.08). The only significant change in the lipid profile was an increase in high-density–lipoprotein cholesterol (50.46 ± 2.34 vs. 55.08 ± 2.51 mg/dL). Leptin levels increased (81.43 ± 7.87 ng/mL to 94.10 ± 6.56 ng/mL), and adiponectin decreased nonsignificantly, resulting in an increased leptin–adiponectin ratio (12.56 ± 1.70 to 15.86 ± 2.24); resistin levels increased (9.37 ± 1.09 ng/mL to 11.72 ± 1.10 ng/mL); and baseline ghrelin levels decreased (701.64 ± 59.79 pg/mL to 581.72 ± 36.07 pg/mL). After tE 2 , no significant changes in IR parameters occurred, except for a decrease in glucose–insulin ratio. There were no changes in lipid parameters. Leptin did not change (72.7 ± 9.3 ng/mL to 78.8 ± 7.9 ng/mL), whereas adiponectin levels showed statistically significant increase (7.97 ± 0.7 μg/mL vs. 9.96 ± 1.1 μg/mL), with no change in the leptin–adiponectin ratio. Resistin levels did not change significantly, and ghrelin levels decreased (888.52 ± 109.98 pg/mL vs. 579.04 ± 39.30 pg/mL). Conclusion(s) This short-term study suggests that oral E 2 may worsen IR and adipocytokine parameters, worsening cardiovascular risk. Transdermal E 2 had minimal effects on IR and resulted in higher adiponectin. Although these data may not reflect alterations that occur with estrogen therapy in more metabolically normal postmenopausal women or with longer term therapy, the findings suggest that tE 2 may be a preferable treatment for obese women with MBS. Long-term studies are needed to make any recommendations.

Pre-mRNA processing is an important mechanism for globally modifying cellular protein composition during tumorigenesis. To understand this process during lung cancer, expression of two key pre-mRNA alternative splicing factors was compared in a mouse model of early lung carcinogenesis and during regenerative growth following reversible lung injury. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and alternative splicing factor/splicing factor 2 (ASF/SF2) act antagonistically to modulate splice site selection. Both hnRNP A1 and ASF/SF2 contents rose in adenomas and during injury-induced hyperplasia compared to control lungs, as measured by immunoblotting. While both proteins increased similarly during compensatory hyperplasia, hnRNP A1 increased to a much greater extent than ASF/SF2 in tumors, resulting in a 6-fold increase of the hnRNP A1 to ASF/SF2 ratio. Immunohistochemical analysis showed that hnRNP A1 localized exclusively within tumor nuclei, while ASF/SF2 appeared in cytoplasm and/or nuclei, depending on the growth pattern of the tumor cells. We also demonstrated cancer-associated changes in the pre-mRNA alternative splicing of CD44, a membrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions. hnRNP A1 and ASF/SF2 expression is thus differentially altered in neoplastic lung cells by mechanisms that do not strictly arise from increased cell division. These changes are influenced by tumor histology and may be associated with production of variant CD44 mRNA isoforms. © 2004 Wiley-Liss, Inc.

Cachexia is a severe muscle-wasting syndrome associated with several chronic diseases such as cancer and AIDS. Muscle mass loss significantly decreases prognosis and survival. The mechanisms of muscle atrophy and the specific proteins targeted for degradation have been intensely studied and are potential therapeutic targets. Published reports that myosin heavy chain (MyHC), the most abundant protein by mass in skeletal muscle, is selectively targeted for degradation in cancer cachexia remain controversial. Here we show that the results of previous studies showing a selective decrease in MyHC are likely an artifact resulting from muscle lysis methods which do not solubilize myosin out of myofibrils. We show that MyHC decreases in parallel with other myofibrillar proteins in cachectic skeletal muscle, which has mechanistic and therapeutic implications. These findings should lead to mechanistic insight into the stoichiometry of sarcomeric disassembly and degradation during cancer cachexia.

Objective: Metabolic syndrome (MBS) is a significant health care problem in postmenopausal women and is driven largely by obesity. We wished to assess the prevalence of insulin resistance (IR), diagnosed using practical methods, and whether several adipocyte factors (adiponectin, leptin, resistin) or the gastric peptide ghrelin, associated with cardiovascular risk, might be abnormal and may relate to IR. Study design: We evaluated 37 obese postmenopausal women with MBS and 34 matched obese premenopausal controls, as well as 14 non-obese premenopausal controls. We measured fasting glucose and insulin, performed 75g 2 hr oral glucose tolerance and intravenous insulin tolerance tests to assess IR, and measured fasting lipids, adiponectin, leptin, resistin and ghrelin. Results: The kinetic decline in glucose after insulin (kITT) as a marker of IR was the most frequently abnormal test (abnormal in 81%), with QUICKI, HOMA, and a modification of the Matsuda-DeFronzo index (ISIM) abnormal in 76, 73, and 68%, respectively. The GIR was abnormal in only 35% of subjects. Leptin and resistin were elevated and adiponectin and ghrelin were decreased in the postmenopausal women, compared to both groups of premenopausal controls. BMI correlated strongly with markers of insulin resistance as well as adipocytokine values. After controlling for BMI, only leptin was predictive of ISIM. Conclusion: Being overweight after menopause results in worsening IR and elevations in adipocytokine levels. While BMI is the most important factor, abnormal adipocytokine secretion may enhance IR and increase cardiovascular risk in postmenopausal women. 2006 Mosby, Inc. All rights reserved.

Studies using transgenic and knockout mice have demonstrated that particular cytokines influence lung tumor growth and identified prostaglandin E2 (PGE2), prostacyclin (PGI2) and nitric oxide (NO) as critical mediators of this process. PGE2 and NO were pro-tumorigenic while PGI2 was antitumorigenic. We describe herein an in vitro experimental approach to examine interactions among cytokines, prostaglandins (PGs) and NO. PGE2, PGI2, and NO levels were assayed in culture media from non-tumorigenic mouse lung epithelial cell lines, their spontaneous transformants and mouse lung tumor-derived cell lines, before or after exposure to the cytokines TNFalpha, IFNgamma and IL1beta, alone and in combination. More PGE2 than PGI2 was produced by neoplastic cells, while the opposite was observed in non-tumorigenic lines. Cytokine exposure magnified the extent of these differential concentrations. The PGE2 to PGI2 ratio was also greater in chemically-induced mouse lung tumors than in adjacent tissue or control lungs, supporting the physiological relevance of this in vitro model. Expression of PG biosynthetic enzymes in these cell lines correlated with production of the corresponding PGs. Cytokine treatment enhanced NO production by inducing the inflammation-associated biosynthetic enzyme, inducible NO synthase (iNOS), but this did not correlate with the neoplastic status of cells. Inhibition of iNOS or cyclooxygenase 2 activity using aminoguanidine or NS-398 respectively, demonstrated that NO did not affect PG production nor did PGs influence NO production. Since lack of iNOS inhibits mouse lung tumor formation, we propose that this is independent of any modulation of PG synthesis in epithelial cells. The similar normal/neoplastic trends in PGE2 to PGI2 ratios both in vitro and in vivo, together with an amplification of this difference upon cytokine exposure, are consistent with the hypothesis that cytokines released during inflammation exacerbate differences in the behavior of neoplastic and normal lung cells.

Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases.