Alice M. Boylan

Co-ordinated over-expression of the MRP and γ-glutamylcysteine synthetase genes, but not MRD1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines

While human malignant mesothelioma is extremely resistant to chemotherapy, its intrinsic resistance mechanisms remain largely unknown. In this study, we used normal human mesothelial cells and 5 human mesothelioma cell lines not previously exposed to chemotherapeutic agents to demonstrate that the mRNA for the multidrug resistance‐associated protein (MRP) and γ‐glutamylcysteine synthetase (γ‐GCSh) heavy subunit genes, but not the P‐glycoprotein (MDR1) gene, are co‐ordinately over‐expressed in mesothelioma cell lines. Expression of MRP as detected with an anti‐MRP antibody correlated with decreased doxorubicin accumulation and resistance of mesothelioma cells to this drug. Our results strongly suggest roles for MRP and γ‐GCSh in chemoresistance in mesotheliomas. Int. J. Cancer 75:757–761, 1998.© 1998 Wiley‐Liss, Inc.

CaSm (LSm-1) Overexpression in Lung Cancer and Mesothelioma Is Required for Transformed Phenotypes

CaSm (cancer-associated Sm-like) was originally identified based on elevated expression in pancreatic cancer and in several cancer-derived cell lines. It encodes a 133-amino acid protein that contains two Sm motifs found in the common snRNP proteins and the LSm (like-Sm) family of proteins. Lung tumors and mesotheliomas express high levels of CaSm mRNA and protein compared with adjacent nontumor and normal lung tissue, measured by immunohistochemistry, qRT-PCR, and Western blot analyses. In addition, several human lung cancer- and mesothelioma-derived cell lines have elevated CaSm expression. Two cell lines, transfected with and expressing antisense CaSm RNA, demonstrate altered transformed phenotypes, reducing their ability to form colonies in soft agar and tumors in SCID mice. Furthermore, RNAi-mediated reduction of CaSm RNA and protein is associated with inhibition of cellular growth. These data support the model that elevated CaSm expression in epithelial tissue contributes to the transformed state. Cell lines expressing exogenous CaSm also exhibit transformed characteristics, including increased anchorage-independent colony formation and tumor growth. Thus, the results of loss of function and gain of function studies presented both indicate that CaSm functions as an oncogene in the promotion of cellular transformation and cancer progression.

Utilizing endobronchial ultrasound with fine-needle aspiration to obtain tissue for molecular analysis: a single-center experience.

Background:Lung cancer is the leading cause of cancer deaths worldwide, with the majority of patients presenting with advanced disease for which surgery is not an option. Recently, a number of genetic mutations have been identified that predict response to chemotherapy, making the acquisition of tissue for molecular analysis important for treatment planning. It has previously been demonstrated that samples obtained using endobronchial ultrasound fine-needle aspiration (EBUS-FNA) are adequate for this analysis. This is the first report on the use of EBUS-FNA specimens for analysis of the epithelial mesenchymal transition (EMT) pathway. Methods:A total of 74 consecutive patients with known or suspected lung cancer undergoing EBUS for diagnosis and/or staging were enrolled in this study. Total RNA was isolated from the FNA specimens, reverse transcribed, and analyzed for expression of a panel of genes associated with EMT using a probe-based real-time quantitative polymerase chain reaction. Results:A total of 150 lymph nodes were sampled from the 74 patients who participated in the study. There was adequate tissue to perform real-time polymerase chain reaction in 130 (86%) of the nodes. Conclusions:EBUS-FNA samples are adequate for analysis of novel markers and pathways, including EMT, which may predict prognosis, responsiveness to therapy, and provide potential targets for new drug development. As the treatment of lung cancer shifts toward a more personalized approach, EBUS-FNA will likely play a central role in tissue acquisition for diagnosis, staging, and molecular analysis.

Development and Pilot of a Checklist for Management of Acute Liver Failure in the Intensive Care Unit

Introduction Acute liver failure (ALF) is an ideal condition for use of a checklist. Our aims were to develop a checklist for the management of ALF in the intensive care unit (ICU) and assess the usability of the checklist among multiple providers. Methods The initial checklist was developed from published guidelines and expert opinion. The checklist underwent pilot testing at 11 academic liver transplant centers in the US and Canada. An anonymous, written survey was used to assess the usability and quality of the checklist. Written comments were used to improve the checklist following the pilot testing period. Results We received 81 surveys involving the management of 116 patients during the pilot testing period. The overall quality of the checklist was judged to be above average to excellent by 94% of users. On a 5-point Likert scale, the majority of survey respondents agreed or agreed strongly with the following checklist characteristics: the checklist was easy to read (99% agreed/agreed strongly), easy to use (97%), items are categorized logically (98%), time to complete the checklist did not interfere with delivery of appropriate and safe patient care (94%) and was not excessively burdensome (92%), the checklist allowed the user the freedom to use his or her clinical judgment (80%), it is a useful tool in the management of acute liver failure (98%). Web-based and mobile apps were developed for use of the checklist at the point of care. Conclusion The checklist for the management of ALF in the ICU was shown in this pilot study to be easy to use, helpful and accepted by a wide variety of practitioners at multiple sites in the US and Canada.

Common Problems in Critically Ill Obstetric Patients, With an Emphasis on Pharmacotherapy

Pharmacological treatment of critically ill obstetric patients can be especially challenging due to the complexity of caring for 2 patients, with a paucity of research to support practice. This review will provide practitioners with primary recommendations for management of the critical illnesses most commonly encountered in pregnancy and will discuss the scientific and clinical merit of these recommendations.

Lack of correlation of MRP and γ-glutamylcysteine synthetase overexpression with doxorubicin resistance due to increased apoptosis in SV40 large T-antigen-transformed human mesothelial cells

Purpose: Evidence suggests that viral proteins such as simian virus large T-antigen (SV40 TAg) play a role in the response of cancer cells to chemotherapeutic agents. In this study, we investigated whether SV40 TAg-immortalized human mesothelial cells express drug resistance-related proteins and display resistance to chemotherapy, and whether SV40 TAg transformation affects apoptosis. Methods: We determined the mRNA and protein levels of the multidrug resistance-associated protein (MRP), γ-glutamylcysteine synthetase heavy subunit (γ-GCSh), and P-glycoprotein (product of the MDR1 gene) by RT-PCR and Western blotting, respectively, in normal human mesothelial (NHM) cell and SV40 TAg-transformed human mesothelial (Met-5A) cells. The effect of increasing concentrations of doxorubicin (DOX) on these cells was investigated using an MTT cytotoxicity assay, and the glutathione (GSH) content was measured spectrophotometrically. DOX accumulation in these cells was measured by treating the cells with [14C]DOX followed by scintillation counting. Cytoplasmic DNA fragmentation due to apoptosis following DOX treatment of the cells was quantitated by ELISA. Results: We showed that the MRP and γ-GCSh genes, but not MDR1, are coordinately overexpressed in Met-5A cells compared with NHM cells. Expression of MRP protein as detected by an anti-MRP antibody correlated with increased GSH levels and decreased accumulation of [14C]DOX in Met-5A cells compared with NHM cells. However, Met-5A cells were twofold more sensitive to DOX than NHM cells. In addition, quantitative measurement of apoptosis when cells were treated with 0.05 and 0.5 μM DOX revealed that drug-induced apoptotic cell death was increased about 1.4- and 3.0-fold, respectively, in Met-5A cells compared with NHM cells. Conclusions: These results suggest that increased levels of apoptosis might help overcome the DOX resistance effects of MRP/γ-GCSh overexpression in SV40 TAg-transformed Met-5A cells.

Abstract 2955: LSm1 over expression contributes to an increase in alternative splicing in lung cancer cells

RNA splicing is a key molecular event that allows for substantial protein diversity. Through this process, a single gene magnifies its coding capacity by expressing several related proteins with diverse and often antagonistic functions leading to cell or tissue specific changes in mRNA through changes in splice site choice. Alternative splicing, which represents the suppression of constitutive splice sites and/or the use of suboptimal sites, is elevated in cancer and many other disease states. Pre-mRNA splicing is coordinated by the spliceosome, a macromolecular ribonuclear complex that assembles on the pre-mRNA as it is transcribed. Due to the number of proteins involved in splicing, the diversity of pre-mRNA substrates and the multi-step process of spliceosome assembly and catalysis, regulation of splicing can occur at any point in the pathway. The LSm proteins are a family of eight RNA binding proteins that form two seven membered ring structures and contribute to mRNA stability and splicing. LSm1 forms a complex with LSm2-7 that targets mRNA for degradation in P bodies. LSm8 also forms a complex with LSm2-7 that binds the U4 U5 U6 tri-SNRP, an integral part of the spliceosome. Previously, it was shown that LSm1 was elevated in many different types of cancer and contributed to the malignant phenotype. Recently, alternative splicing has been shown to contribute to the activation of oncogenes and the inactivation of tumor suppressor genes. To determine if the over expression of LSm1 could contribute to alternative splicing, a well differentiated lung cancer cell line was infected with adenovirus expressing the open reading frame of LSm1 or GFP. The RNA from these cells was analyzed for alternative splicing using Agilent SpliceArray™ microarray slides. Significant differences were seen between the cells treated with control virus and the LSm1 expressing virus in the splicing patterns of several genes that could contribute to the cancer phenotype, including hepatoma derived growth factor (HDGF), CD44 and c-myc. Thus, over expression of LSm1 leads to changes in alternative splicing that impacts the malignant phenotype of cancer cells. 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 2955.

Abstract 3305: Markers of epithelial-mesenchymal transition as indicators of prognosis in lymph node aspirates from lung cancer patients

Lung cancer accounts for the largest number of cancer-related deaths worldwide. Despite a new more discriminatory staging system for predicting survival, great variation in outcome within stage persists. Gene expression arrays and microRNA signatures from surgically resected primary tumors can predict survival and drug sensitivity within stage I disease better than standard staging protocols. Genes associated with epithelial mesenchymal transition (EMT) contribute to the malignant phase of tumor cell growth by controlling the switch to a metastatic phenotype. We hypothesize that the analysis of expression of EMT markers in thoracic lymph nodes may predict survival outcomes and drug responses in lung cancer patients. Endobronchial ultrasound fine needle aspiration (EBUS-FNA) of lymph nodes was performed on patients with known or suspected lung cancer to diagnose and/or stage disease. Total RNA was isolated from the FNA specimens, reverse transcribed, and analyzed for expression of a panel of genes associated with EMT using a probe-based real-time quantitative polymerase chain reaction (pqRT-PCR). Samples were analyzed for the expression of the epithelial markers cytokeratin 17 (CK17) and cytokeratin 19 (CK19) and the EMT markers Ecadherin (ECAD), Ncadherin (NCAD), and transcription factors snail, slug, ZEB1 and ZEB2. Samples from thirty-seven patients were processed for the study. Patients with metastatic disease had higher levels of snail and NCAD and lower levels of slug and ZEB2 compared to node negative and node positive patients, while patients with node negative disease had higher slug and lower levels of NCAD compared to node positive patients. ECAD did not vary by node positivity or presence of metastasis. The patients with adenocarcinoma on cytology showed higher levels of ECAD and snail, while patients diagnosed with squamous cell carcinoma had higher levels of ZEB1. In contrast, two patients with benign disease had low levels of all markers. Four patients with cytopathologically negative nodes expressed the epithelial markers CK17 and CK19, which are not typically present in lymph nodes. In limited follow-up, two patients with cytopathologically negative lymph nodes but high levels of ZEB1 had progression of disease. From this pilot study we have shown that EBUS-FNA consistently provides adequate samples from which RNA can be isolated and that genes associated with EMT can be used to identify cancer cells that have migrated to adjacent lymph nodes. We plan to enroll a total of 100 patients and follow them for 2 years to determine if this technique can identify patients at higher risk of metastatic disease. 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 3305.