NOTE: Start/end dates changed to 3/31/2005-3/31/2010 per grant documents from PI (12/06)

The NSCOR has 4 Projects:

1. Genetic and epigenetic changes in human bronchial epithelial cells following exposure to HZE particle irradiation;

2. Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells;

3. Effect of HZE particle irradiation on functional progression of human lung cancer at the cellular and organotypic level; and

4. Effects of HZE particles on the development of lung cancer in vivo in novel mouse models.

These projects are supported by 4 Cores: Administrative; Cell Culture; Genomics and Proteomics; and Biostatistics and Bioinformatics. In addition, with funding from the Department of Energy (DOE) contributing to this NSCOR, studies of low-LET and low dose radiation in the same model systems are also being undertaken. The HZE particle irradiation is done at Brookhaven National Lab, and the biostatistical analysis shows the experiments are powered to help achieve NASA mandated risk estimate confidence levels. Finally, since this group has substantial UTSW institutional commitments, holds a NCI Special Program of Research Excellence (SPORE) in Lung Cancer, a Center for the NCI Early Detection Research Network (EDRN) program, and is part of the NCI sponsored Genetic Epidemiology of Lung Cancer Consortium (GELCC) there is great synergism and additional resources available for the successful completion of this proposal.

Overall we are conducting research to investigate how biological endpoint and systems biology approaches can be used to integrate research on the cellular and molecular as well as tissue level of radiation damage that will lead to significant reductions in the uncertainties in risk projection models of lung cancer. As will be described in this report, we are confident and will provide compelling evidence that the sum of the efforts of our assembled team are significantly contributing more to assessing cancer risk associated with space radiation compared to the individual components.

Specific Aim 1: To quantitate the effects of HZE particle irradiation in causing genetic changes in HBECs and their isogenic derivatives.

Specific Aim 2. To quantitate the amount of epigenetic changes caused by HZE particle irradiation in HBECs and their isogenic derivatives using high throughput quantitative methylation specific PCR (MSP) assays for acquired promoter methylation

Specific Aim 3. To quantitate the effects of HZE particle irradiation in causing expression profile (mRNA and protein) changes in human bronchial epithelial cells (HBECs) and their isogenic derivatives containing various lung cancer-related mutations using array based genome wide and multi protein detecting approaches.

Specific Aim 4. To determine the effect of inter-individual variation on the effects of HZE particle irradiation in causing genetic changes, epigenetic changes, mRNA and protein expression profile changes in human bronchial epithelial cells (HBECs).

Project 1: In the last year this project has focused on 3 specific areas. mRNA Expression Analysis. Comparative gene (mRNA) expression analysis using genome wide array technology at equi-toxic doses of low LET gamma radiation vs. that of HZE radiation (28Si and 56Fe) at different time points. We find that there are different mRNA expression profiles generated by each of the different types of radiation and that this is also dependent on the oncogenotype of the HBECs radiated. DNA Methylation Profiles. While there are no global methylation changes we are exploring genome wide changes using array technology as well as quantitative methylation specific PCR (MSP). miRNA Expression Profiling. We are using genome wide array technology to profile miRNA changes after low and high LET radiation and low dose gamma radiation. Project 1 is now close to hitting the milestones described in the original application. We will very shortly have a set of well characterized genomic responses (both mRNA, DNA copy number, DNA methylation, and miRNA expression) to low LET and HZE radiation (Si, Fe) exposures in a series of isogenic immortalized human bronchial epithelial cells (HBECs) which differ only by specific oncogenic changes introduced (e.g. KRAS, p53, EGFR, E6, E7). These variants thus include cells with altered p53, RAS and EGFR signaling. Our goal is to mine the data available in order to develop biomarkers of response that may be used for relative risk determinations of lung cancer from space radiation exposure. Determining such biomarkers dovetails with the transformation studies of Project 3. With the Biostatistics/Bioinformatics Core (Core D) e are using multiple analysis methodologies to develop biomarkers including Gene Set Enrichment, pathway analysis, smar, and Bayesian analysis for interaction nodes. These biomarkers are also being evaluated in mouse models in Project 4 and can be used to address inter-individual differences in carcinogenic response for risk determination.

Project 2: Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells. David Chen, Ph.D. Project Lead, Sandeep Burma, Ph.D. Co-Lead.

Specific Objectives: Aim 1. To test the hypothesis that the immediate cellular response to DNA damage induced by HZE would be different and more complex than that induced by X-rays.

Aim 2. To test the hypothesis that persistent DNA damage induced by HZE is more complex and difficult to repair than that induced by X-rays and, therefore, more deleterious.

Aim 3. To determine if damage induced by HZE is preferentially repaired by non homologous end joining (NHEJ) or homologous recombination (HR) or is preferentially repaired in certain phases of the cell cycle.

Aim 4. To compare the response of human bronchial epithelial cells (HBECs) as non immortalized, immortalized, and with premalignant oncogenic changes in 3D cultures to HZE exposure using a high throughput approach.

Project 2: This project has had two major developments this year. First development of human lung epithelial cells with endogenous EGFP labeled 53BP1 to allow live cell imaging of DNA repair after low and high LET Radiation in both 2 dimensional (2D) and 3 dimensional (3D). To investigate the cellular responses to DNA double strand breaks (DSBs) induced by low- and high-LET irradiation (IR) in vivo, we generated a human bronchial epithelial cell (HBEC3) line that stably expresses near-physiological levels of EGFP-53BP1 and used this human lung epithelial cell line in a three-dimensional (3D) model tissue culture system (Matrigel) to study induction and repair of DNA DSBs under conditions that were close to those in 3D tissues in vivo by monitoring the formation of 3BP1 foci development and resolution with by monitoring the EGFP labeled endogenous protein. We found that survival curves were identical for HBECs with and without the 53BP1 expression construct. Before we used this system for DSB repair study, we extensively characterized these cells after growing them in Matrigel. These analyses indicated that when we grow HBECs in this extracellular matrix (ECM) culture, they attain a growth-arrested state at day 6, similar to 3D cultures of mammary epithelial cells.

Second, use of HEBECs with endogenous EGFP-53BP1 to study foci dissolution kinetics data after different types of radiation. These studies indicated that a significant proportion of iron-ion-induced damage was irreparable both in 2D and 3D structures, but the number of persistent foci was significantly higher in 3D structures. Collectively, these EGFP-53BP1 foci dissolution analyses suggest that the DNA DSBs induced by high-LET IR are differentially repaired in 2D and 3D structures.

Project 3. Effect of HZE Particle Irradiation on Functional Progression of Human Lung Cancer. John D. Minna, M.D. Project Lead, Jerry Shay, Ph.D. Co-Lead.

Specific Aim 1. Effects of HZE particles on early events in lung cancer progression (soft agar colony formation and other markers of cellular transformation).

Specific Aim 2. Effects of HZE particles on intermediate events in lung cancer progression (invasion in 3 D organotypic cultures).

Specific Aim 3. Effects of HZE particles on late events in lung cancer progression (ability to form tumors in immune deprived mice).

Project 3: We have made substantial progress in the determination of transformation by HZE particles in normal human bronchial epithelial cells (HBEC3KT). (Transformation as defined by the ability to grow in soft agar.) We last reported that there was a peak of transformation (two logs higher from a base rate of 10-7 to ~10-5) for 56Fe particle irradiations at 0.25 Gy which then diminished with higher doses and was more marked than the increase see with gamma radiation at 2-4 Gy. These data are updated in with the addition of 28Si data, which also shows the same response. Interestingly, the HBEC3KT Kras/p53 cell line (which has a four log higher spontaneous transformation rate (10-3) did not show any increase in transformation rate by gamma, Si, or Fe up to 1 Gy. We have now isolated over 160 individual transformed foci and these show that ~50% of the clones breed true for this phenotype. We have studied these and found that they exhibit epithelial to mesenchymal transition (EMT) compared to non irradiated control transformed foci by both morphology and molecular markers (vimentin, ecadherin expression). In addition, they have an altered radiation survival curve phenotype. With these cell isolates in hand we will begin to determine tumorigenicity. Tumors themselves will be examined for radio sensitivity and aggressiveness. Tumor phenotype will be compared to those mouse lung carcinomas generated after Fe-exposure in Project 4, which were characterized as especially aggressive. Those cells that develop tumors will be characterized at the genomic and proteomic level and compared against the phenotype of human lung tumors generated via smoking or tumors seen in non-smokers, or via radiation-exposure, in particular via inhalation of radioisotopes such as Pu or Rn. This should allow the characterization and risk for radiation-induced lung cancers, particularly those induced by HZE exposure.

Project 4. Effects of HZE Particles on the Development of Lung Cancer in Novel Mouse Models. Jerry W. Shay, Ph.D. Project Lead, James Richardson, DVM, Ph.D. Co-Investigator, Oliver Delgado, Student Assistant.

Specific Aim 1. Use the K-RAS transgenic mouse model of human lung cancer to determine effects of low- and high-LET radiation in tumor initiation and progression.

Specific Aim 2. Use additional transgenic mouse models to determine the effects of low- and high-LET radiation in both living animals and in isolated tissues.

Summary of Project 4: 1. Fractionated and single dose irradiation of 1Gy 56Fe- particles do not increase tumor incidence

2. Tumors in the LA1 K-RAS mice irradiated with fractionated doses of (0.2Gy/day, total 1 Gy (1GeV/n) 56Fe- particles have a statistically significant increased probability of progressing to invasive carcinoma in comparison to a single acute 1Gy 56Fe- dose.

3. These results suggest significant effects of fractionated high-LET irradiation on cancer progression

4. To test this hypothesis we have initiated additional single dose and fractionated doses to confirm these important observations. We have irradiated mice with fractionated x-ray (0.6 Gy/day x 5 days) and have not observed an increase incidence of invasive tumors. We have initiated fractionated 56Fe- particle experiments (0.1 Gy/day x 10 over a 12 day period).

5. We are testing the hypothesis that fractionated high-LET irradiation induces persistent inflammatory signaling that does not occur in single doses. An alternative hypothesis is that there could be a role of stem cell repopulation for lung tissue that is important in cancer progression. Thus, acute doses of high-LET ions may increase cell killing, reducing the number of target stem cells available for initiation and progression to lung cancer.

6. We have provided non lung tissues from our control and irradiated mice to other investigators (Ameila Eisch, brain tissue; Zhi-Min Yuan, kidney, liver, spleen)

7. Mouse telomerase knockout model of lung cancer. Our rationale and overall goal is to produce a mouse with “humanized” telomere length so that we can irradiate with X-ray, proton and HZE particles to determine genomic changes and DNA damage/repair in the lung and other tissues. Inbred strains of mice have extraordinarily long telomeres and thus much of the genomic instability due to telomeres shortening in humans is unlikely to be observed in typical mouse irradiation studies. We have reached the third generation of breeding mTERT KO to KO to obtain short (more human like) telomere length and initiated our first experiments in the Fall 2009 NSRL run. These animals have been returned from the BNL and we will be conducting pilot microarray experiments to compare to similarly irradiated wild-type mice for DNA damage and inflammatory changes that may distinguish mice with humanized telomeres from typical inbred strains of mice. Future experiments will be initiated to determine if more end joining, end-end fusions, cytogenetic alterations, micronuclei, ongoing reactive oxygen species (ROS) occur when these mice are exposed to low- and high-LET irradiation.

8. EGFR tyrosine kinase domain mutation mouse/ Our rationale for initiating experiment with a new mouse model is that astronauts do not smoke. EGFR mutations are frequently found in lung cancers from never smokers. The models we have chosen to use have an EGFR mutation or small deletion that can be induced by adding doxycycline in the food or drinking water. We now have the mice in our breeding colony and have been scaling up breeding pairs. Experiments under consideration when sufficient numbers of animals are available include the following: We will activate the EGFR-mut about a month prior to low- and high-LET irradiation to compare DNA damage and repair in tumor versus normal tissue; We will also subject these animals to low- and high-LET irradiation prior to activating the EGFR mutation and then add doxycycline to the food for three months. These experiments are designed to further dissect stromal/epithelial differences in cancer progression. The hypothesis that we propose to test is that by activating the stromal compartment prior to tumor initiation the host environment may be permissive for the future growth of the tumors; We will compare the EGFR-mut inducible model to the inducible K-RAS mouse model.

9. DOE Related studies: One aspects of our NSCOR is supported by the DOE. While low doses or even low dose rates have not yielded informative animals results in most instances, we have now formulated a series of testable hypothesis that may be tenable. We propose to send both the mutant EGFR and K-RAS mice to the Colorado State University (CSU) low dose facility and then expose them to low dose rates (10 cGy/day) for 10, 20 and 30 days then activate mutant EGFR or K-RAS. We will be able to address the central and unanswered question if tumors appear faster upon activation of the oncogene after low dose exposure to the stroma). We can also determine if activated tumor will or will not regress when the doxycycline food is removed at 90 days.

Core A: Administrative Core. Leads: John Minna and Jerry Shay, and UTSW NSCOR Administrator: Brenda Zielke.

The Administrative Core has established accounts and control over disbursement of all funds from this NSCOR application. It has also kept track of all personnel employed by this NSCOR. It has provided a central place for communication and coordinated data sharing In addition, it has established a series of biweekly to monthly scientific and planning meeting of the NSCOR investigators at UTSW. Approximately 25 scientific personnel attend each of these meetings. At each meeting administrative matters are discussed, experiments are planned, administrative changes made by the senior leadership team, and most importantly a scientific presentation by one of the NSCOR investigators is made and critiqued by the other investigators. Dr. Minna serves as the Administrative Core Lead with Dr. Jerry Shay as the Administrative Core Co-Lead. The Administrative Core coordinated all of the arrangements for the participation of several NSCOR investigators for the NASA reverse site visit February 18-19, 2009, and travel for several investigators to the 20th NASA investigators meeting in Cologne Germany. The Administrative core also arranged the Internal Scientific Advisory Board Review (March 4, 2008 as part of a mini-retreat at UT Southwestern) and the External Advisory Board Review (October 15, 2008 Mary Helen Barcellos-Hoff, Robert Ullrich and Martin Brown participating). Finally, the Administrative Core coordinated travel of multiple personnel to BNL for experimental work.

Core B. Cell Culture. Core Leads: John D. Minna, M.D. and Jerry Shay, Ph.D.

Enhancement of human bronchial epithelial cell 3D lung model of tissue like development. There is increasing evidence that cancer arises from stem cell populations that are important for the normal turnover of tissues and the rapid repair of tissues in instances of injury. In the lung there is evidence for a bronchiole alveolar stem cell (BASC) population that may give rise to lung carcinoma. We developed a panel of immortalized human bronchial epithelial cells and have previously demonstrated that these cells when placed in an air medium interface overlying a fibroblast stromal collagen matrix could differentiate into mucous and ciliated bronchial epithelial cells. These results were expected since the cells originated from the central airways and were obtained from mid size bronchial tissues. We examined these cells for a variety of differentiated cell markers and observed that they expressed p63 (a transit amplifying cell marker) and surprisingly surfactant protein A (SP-A), CC10, a Clara cell marker, and when differentiated weak expression of the thyroid transcription factor TITF1 (known to be important in adenocarcinomas of the peripheral airway of the lungs). These peripheral markers associated with Clara and type II alveolar cells imply that our immortalized HBECs have enhanced plasticity and should be considered pluripotent lung stem cells. We next placed these cells into Matrigel overlying a lung fibroblast (IMR90) feeder layer and observed that the cells could efficiently differentiate into spheroids or cyst like structures. Studies of these HBECs growing in Matrigel and forming cysts and SACs indicated they started to express SP-A and morphologically resembled small bronchioles and alveolar sacs. When examined in the electron microscope, these cells also had lamellar bodies which are a hallmark of type II alveolar cells. This new improved 3D model of lung cell differentiation has many advantages over the previous model. First, they can differentiate in 3-5 days in vitro making it feasible to set these up at BNL for future NSRL runs. This is in contrast to the air liquid interface for differentiation into ciliated and mucous secreting bronchial cells which require a minimum of two weeks of culture and our previous experience indicated that they do not ship well to BNL. Second, these differentiating small airways cells resemble the type of lung cancer that is likely to be increased in astronauts on long-term space missions. Thus, during the 2008 and 2009 NSRL runs we initiated our irradiation experiments with these cells (see Project 2 overview). Importantly, we introduced a reporter gene 53BP1 containing EGFP so that they sites of irradiation induced damage could be visualized in real time. One final improvement in the 3D culture of our immortalized HBECs consists of culturing them on top of Matrigel. Under these conditions, the cells form web like structures then bud as if they are formed alveoli. This new method also permits a way to have 3D cultures in 5 days and thus promises to be a new addition to our arsenal of ways to examine the influence of irradiation on lung cells under various conditions.

Core C. Genomics and Proteomics Core. Core Lead: Michael Story, Ph.D., Ching-Reng Yang, Ph.D. Core Co-Lead.

Genomics Core: The Core has continued to perform genome wide profiling including mRNA, miRNA, DNA SNP copy number, and DNA methylation, as well as reverse phase protein array (RPPA) profiling on samples before and after radiation in support of all of the projects of this NSCOR. In addition, this Core interfaces with the Biostatistics and Bioinformatics/Database Core (Core D) to ensure the primary results from these genome wide studies are available for appropriate biostatistical and bioinformatics analysis. These are very precise technologies that requires extensive experience to perform and sophisticated instrumentation to carry out. Furthermore, having a single facility through which all array analysis passes minimizes the variability brought about by different individuals processing samples even though the same platform is being used. For these reasons we included a Core facility (Core C, Genomics and Proteomics) in this NSCOR grant. This NSCOR Core also interfaces with the UTSW Cancer Center Microarray and the UTSW school wide Cores.

The NSCOR Core has the following duties: 1. Interact with Project Leaders and investigators in the design of experiments that use microarray technologies (mRNA, miRNA, DNA copy number, and proteomics) in their experimental approach. Biostatistics and bioinformatics will also be integrated in this process. 2. Establish procedures for consistency in sample preparation and RNA, DNA, and protein extraction, test all RNA, DNA, and protein samples for integrity, and provide reports to that effect. 3. Perform all hybridizations, perform all scans, and place data on a secure server. 4. Provide early analysis on array quality control and preliminary data analysis to investigators. 5. Interact with the Biostatistics and Bioinformatics Core (Core D) to provide bioinformatic analysis of data according to project requirements.

NSCOR Core D: Biostatistics and Bioinformatics. Core Lead: Xian-Jin Xie, Ph.D.; Core Co-Lead: Luc Girard, Ph.D. and Yang Xie, Ph.D.

The Biostatistics and Bioinformatics Core (Core D) provides comprehensive biostatistics and bioinformatics expertise to ensure the statistical integrity and to optimize data analysis of the University of Texas Southwestern (UTSW) NSCOR studies. In doing so, Core D assists investigators with testable hypothesis formulations and proper planning of experiments, manages experimental data generated by NSCOR projects, conducts and advises data analysis, and assists in interpreting final results. To play an integrated role and to serve all the NSCOR projects in a timely manner, Core D biostatisticians Drs. Xian-Jin Xie, Luc Girard, Yang Xie, Chul Ahn and Song Zhang have participated in all monthly project meetings, ensuring that proper biostatistics and data management considerations are adequately taken during all phases of planned experiments. In conjunction with the Genomics (Expression Profiling and Proteomics) Core (Core C) and the Cell Culture Core (Core B), the Biostatistics and Bioinformatics Core (Core D) has the following specific aims:

- To provide database support and expertise for the collection, storage, integration, and retrieval of all sorts of data generated from NSCOR projects.

- To provide valid and optimal statistical design and to conduct proper analysis required to address the specific aims of each project.

- To review background and rationale, to assist in the design, evaluation, and analysis of new research proposal arising from the individual projects.

- To assist the other Cores and Projects in the proper analysis and interpretations of different types of bioassays, particularly mega-data such as those generated from gene expression arrays.

- To assist in manuscript preparation and to review scientific submissions.

- To assist the NSCOR team in building radiation cancer risk models.

As we progress on all the NSCOR projects, significant amount of new data have been generated since our 2008 annual progress report. As a result of Core D effort, a comprehensive data management system has been established that allows efficient access and sharing of all NSCOR data. Data Collection Close to 10,000 samples (including cell pellets and cryogenically preserved cell lines) and experiments (irradiation, microarrays, mouse data) have so far been collected and stored in the NSCOR database. These data can be viewed or edited by any participating investigator provided they have the necessary permissions.

NASA human research program investigators’ workshop, February 2009. , Feb-2009

Low-dose radiation research investigators' workshop VIII, Bethesda, Maryland, April 2009. , Apr-2009

55th Annual Radiation Research Society meeting, Savannah, Georgia, October 4-7, 2009. , Oct-2009

55th Annual Radiation Research Society meeting, Savannah, Georgia, October 4-7, 2009. , Oct-2009

Radiation Biology and Radiation Protection, Fudan University, Shanghai, China, October 14-16, 2009. , Oct-2009

IASLC conference (International Association for the Study of Lung Cancer), San Francisco, CA, July 31-August 4, 2009. , Aug-2009

Cold Spring Harbor Laboratory annual meeting, Cold Spring Harbor, NY, May 2009. , May-2009

Heavy Ions in Therapy and Space Symposium 2009, Cologne, Germany, July 6-10, 2009. , Jul-2009

IASLC conference (International Association for the Study of Lung Cancer), San Francisco, CA, July 31-August 4, 2009. , Aug-2009

Our studies are providing quantitative data following high-LET irradiation on these HBECs. The primary benefits from this work will be the development of quantitative risk assessment models for astronauts of high-LET radiation in space in order to reduce the uncertainties in prediction of risk of astronauts developing lung cancer as part of long-term missions in space However, the results will also have major impact and benefit to life on Earth includes new knowledge of the effects on human lung epithelial cells of irradiation in terms of quantitative genetic and epigenetic changes and gene expression changes following irradiation which are of potential importance as markers of radiation exposure that could occur through environmental exposures, accidentally or through terrorism. As part of this will be the quantitative response of DNA repair pathways to radiation including specific DNA repair components such as DNA repair enzymes and signaling pathways in lung epithelial cells. We are also determining quantitative changes in biologic phenotypes of lung epithelial cells in response to radiation which include colony formation in liquid and soft agar (anchorage independent growth) and differentiation and invasion in 3 dimensional cultures, and finally growth in vivo as either xenografts (for human cells) or endogenous lung cancers (for mouse models of lung cancer). From all of the above information we will identify novel molecular biomarkers (mRNA, DNA, protein) for lung carcinogenesis. Finally, we are also learning about inter individual variation of the response of HBECs to radiation with respect to the various genetic, epigenetic, mRNA and protein expression, and signaling pathway changes. Our large HBEC panel coupled with new high density single nucleotide polymorphism (SNP) analysis will allow describing polymorphic differences in such responses.

Our lung cancer NSCOR is addressing the following specific critical path roadmap and gap questions:

•We are improving the understanding of space radiation in lung cancer initiation, promotion and progression spanning cell and molecular biology at the cell, tissue and system biology level that will lead to significant reduction in the uncertainties in risk projection models

•We are investigating the increased risk from space radiation as a function of age of exposure, age, latency, gender, tissue, radiation quality and dose rate?

•We are developing tissue specific risk models using human 3D culture and animal models for lung cancer

•We are investigating how aberrant DNA damage processing, genomic instability, epigenetic effects including methylation, persistent oxidative damage, altered senescence, and non-targeted effects contribute to irradiation-associated lung cancer using a variety of surrogate endpoints that are being validated.

Overall we are conducting research to investigate how biological endpoint and systems biology approaches can be used to integrate research on the cellular and molecular as well as tissue level of radiation damage that will lead to significant reductions in the uncertainties in risk projection models of lung cancer. As will be described in this report, we are confident and will provide compelling evidence that the sum of the efforts of our assembled team are significantly contributing more to assessing cancer risk associated with space radiation compared to the individual components.

Specific Aim 1: To quantitate the effects of HZE particle irradiation in causing genetic changes in HBECs and their isogenic derivates.

Specific Aim 2. To quantitate the amount of epigenetic changes caused by HZE particle irradiation in HBECs and their isogenic derivates using high throughput quantitative methylation specific PCR (MSP) assays for acquired promoter methylation

Specific Aim 3. To quantitate the effects of HZE particle irradiation in causing expression profile (mRNA and protein) changes in human bronchial epithelial cells (HBECs) and their isogenic derivates containing various lung cancer-related mutations using array based genome wide and multi protein detecting approaches.

Specific Aim 4. To determine the effect of inter-individual variation on the effects of HZE particle irradiation in causing genetic changes, epigenetic changes, mRNA and protein expression profile changes in human bronchial epithelial cells (HBECs).

Project 1: In the last year this project has focused on 3 specific areas. mRNA Expression Analysis. The first area is comparative gene (mRNA) expression analysis at equi-toxic doses of low LET radiation vs that of HZE radiations, specifically 56Fe. DNA Methylation Profiles. The second area of emphasis this last year was the analysis of radiation-induced changes in DNA methylation patterns. miRNA Expression Profiling. The third area of emphasis dovetails with our gene expression analysis, and represents a relatively new area of research for radiation exposures.

Project 2: Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells. David Chen, Ph.D. Project Lead, Sandeep Burma, Ph.D. Co-Lead.

Specific Objectives :

Aim 1. To test the hypothesis that the immediate cellular response to DNA damage induced by HZE would be different and more complex than that induced by X-rays.

Aim 2. To test the hypothesis that persistent DNA damage induced by HZE is more complex and difficult to repair than that induced by X-rays and, therefore, more deleterious.

Aim 3. To determine if damage induced by HZE is preferentially repaired by non homologous end joining (NHEJ) or homologous recombination (HR) or is preferentially repaired in certain phases of the cell cycle.

Aim 4. To compare the response of human bronchial epithelial cells (HBECs) as non immortalized, immortalized, and with premalignant oncogenic changes in 3D cultures to HZE exposure using a high throughput approach.

Summary of Current Progress/Detailed Results

1. Damage Responses of HBECs Irradiated with Fe Particles.

2. Repair kinetics of DNA damage induced by Fe particles in HBECs

3. DNA damage responses in organotypic 3D cultures of HBECs.

4. Establishment of a YFP-53BP1 system for live cell imaging of DNA damage and its repair:

5. Recruitment of YFP-53BP1 to the sites of DNA DSB in living cells is dose-dependent:

6. Live cell imaging of repair of DNA DSBs induced by gamma-radiation:

7. Expression of EGFP-53BP1 in HBEC3KT cells:

8. EGFP-53BP1 forms foci in 3D cultures of HBEC3KT cells.

9. To compare the ability of monolayer and 3D cultures of HBECs to carry out repair of DSBs induced by gamma rays or HZE by live cell imaging.

Project 3. Effect of HZE Particle Irradiation on Functional Progression of Human Lung Cancer. John D. Minna, M.D. Project Lead, Jerry Shay, Ph.D. Co-Lead.

Specific Aim 1. Effects of HZE particles on early events in lung cancer progression (soft agar colony formation and other markers of cellular transformation).

Specific Aim 2. Effects of HZE particles on intermediate events in lung cancer progression (invasion in 3 D organotypic cultures).

Specific Aim 3. Effects of HZE particles on late events in lung cancer progression (ability to form tumors in immune deprived mice).

Immediate and long-term plans:

Project 1 is now very close to hitting the milestones described in the original application. We will very shortly have a set of well characterized genomic responses (both gene and miRNA expression) to low LET and HZE radiation exposures in a series of isogenic immortalized human bronchial epithelial cells (HBECs) which differ only by specific oncogenic changes introduced (e.g. KRAS, p53, EGFR, E6, E7). These variants thus include cells with altered p53, RAS and EGFR signaling. Our goal will be to mine the data available in order to develop biomarkers of response that may be used for relative risk determinations of lung cancer from space radiation exposure. Determining such biomarkers dovetails with the transformation studies of Project 3. We will use multiple analysis methodologies to develop biomarkers including Gene Set Enrichment, pathway analysis, smar, and in particular, Bayesian analysis for interaction nodes. In future studies these biomarkers can be evaluated in mouse models and in particular, can be used to address inter-individual differences in carcinogenic response for risk determination.

Project 4. Effects of HZE Particles on the Development of Lung Cancer in Novel Mouse Models. Jerry W. Shay, Ph.D. Project Lead, James Richardson, DVM, Ph.D. Co-Investigator, Oliver Delgado, Student Assistant.

Specific Aim 1. Use the K-RAS transgenic mouse model of human lung cancer to determine effects of low- and high-LET radiation in tumor initiation and progression.

Specific Aim 2. Use additional transgenic mouse models to determine the effects of low- and high-LET radiation in both living animals and in isolated tissues.

Specific Aim 1. Use the K-RAS transgenic mouse model of human lung cancer to determine effects of low- and high-LET radiation in tumor initiation and progression.

Summary:

1. Fractionated and single dose irradiation of 1Gy 56Fe- particles do not increase tumor incidence

2. Tumors in the LA1 K-RAS mice irradiated with fractionated doses of (0.2Gy/day, total 1 Gy (1GeV/n) 56Fe- particles have a statistically significant increased probability of progressing to invasive carcinoma in comparison to a single acute 1Gy 56Fe- dose.

3. These results suggest significant effects of fractionated high-LET irradiation on cancer progression

4. To test this hypothesis we have initiated additional single dose and fractionated doses to confirm these important observations. We have irradiated mice with fractionated x-ray (0.6 Gy/day x 5 days) and have not observed an increase incidence of invasive tumors. We have initiated fractionated 56Fe- particle experiments (0.1 Gy/day x 10 over a 12 day period).

5. We are testing the hypothesis that fractionated high-LET irradiation induces persistent inflammatory signaling that does not occur in single doses. An alternative hypothesis is that there could be a role of stem cell repopulation for lung tissue that is important in cancer progression. Thus, acute doses of high-LET ions may increase cell killing, reducing the number of target stem cells available for initiation and progression to lung cancer.

6. We have provided non lung tissues from our control and irradiated mice to other investigators (Ameila Eisch, brain tissue; Zhi-Min Yuan, kidney, liver, spleen)

Core A: Administrative Core. Leads: John Minna and Jerry Shay, and UTSW NSCOR

Administrator: Brenda Zielke.

The Administrative Core has established accounts and control over disbursement of all funds from this NSCOR application. It has also kept track of all personnel employed by this NSCOR. It has provided a central place for communication and coordinated data sharing In addition, it has established a series of biweekly to monthly scientific and planning meeting of the NSCOR investigators at UTSW. Approximately 25 scientific personnel attend each of these meetings. At each meeting administrative matters are discussed, experiments are planned, administrative changes made by the senior leadership team, and most importantly a scientific presentation by one of the NSCOR investigators is made and critiqued by the other investigators. Dr. Minna serves as the Administrative Core Lead with Dr. Jerry Shay as the Administrative Core Co-Lead. The Administrative Core coordinated all of the arrangements for the participation of several NSCOR investigators (Minna, Shay, Chen, Story, Roig, Burma, Delgado) in the 19th NSCOR Investigators Workshop held in Philadelphia, PA June 30-July 2, 2008. The Administrative core also arranged the Internal Scientific Advisory Board Review (March 4, 2008 as part of a mini-retreat at UT Southwestern) and the External Advisory Board Review (October 15, 2008 Mary Helen Barcellos-Hoff, Robert Ullrich and Martin Brown participating).

Core B. Cell Culture. Core Leads: John D. Minna, M.D. and Jerry Shay, Ph.D.

Enhancement of human bronchial epithelial cell 3D lung model of tissue like development There is increasing evidence that cancer arises from stem cell populations that are important for the normal turnover of tissues and the rapid repair of tissues in instances of injury. In the lung there is evidence for a bronchiole alveolar stem cell (BASC) population that may give rise to lung carcinoma. We developed a panel of immortalized human bronchial epithelial cells (HBECs) and have previously demonstrated that these cells when placed in an air-tissue culture medium interface overlying a fibroblast stromal collagen matrix could differentiate into mucous and ciliated bronchial epithelial cells. These results were expected since the cells originated from the central airways and were obtained from mid size bronchial tissues. We examined these cells for a variety of differentiated cell markers and observed that they expressed p63 (a transit amplifying cell marker) and surprisingly surfactant protein A (SP-A), CC10, a Clara cell marker, and when differentiated weak expression of the thyroid transcription factor TITF1 (known to be important in adenocarcinomas of the peripheral airway of the lungs). These peripheral markers associated with Clara and type II alveolar cells imply that our immortalized HBECs have enhanced plasticity and should be considered pluripotent lung stem cells. We next placed these cells into matrigel overlying a lung fibroblast (IMR90) feeder layer and observed that the cells could efficiently differentiate into spheroids or cyst like structures. Studies of these HBECs growing in matrigel and forming cysts and SACs indicated they started to express SP-A and morphologically resembled small bronchioles and alveolar sacs. When examined in the electron microscope, these cells also had lamellar bodies which is a hallmark of type II alveolar cells.

Core C. Genomics and Proteomics Core. Core Lead: Michael Story, Ph.D., Ching-Reng Yang, Ph.D. Core Co-Lead.

Genomics Core: The Core has made two advances in technology that should be described. The first was the analysis of miRNA in the HBEC series. The data collected to date are unique to the radiation community at large. The second area of progress has to do with the generation by Illumina Corp of a new gene expression platform that will drive the price of expression analysis down by half. We are implementing this new platform. This is the second 50% reduction in costs associated with gene expression during the life of this current application. This has already allowed the incorporation of new analytic techniques, miRNA analysis, and with the second cost reduction should allow us the flexibility to expand our analysis further or into new areas of genomics and proteomics.

A. Specific Functions of the Genomics Core

Microarray expression profiling, genome wide DNA copy number (array CGH), and array based protein expression technologies are maturing. However, they are very precise technologies that requires extensive experience to perform and sophisticated instrumentation to carry out. Furthermore, having a single facility through which all array analysis passes minimizes the variability brought about by different individuals processing samples even though the same platform is being used. For these reasons we include a Core facility (Core C, Genomics and Proteomics) in this NSCOR grant. This NSCOR Core interfaces with the UTSW Cancer Center Microarray and the UTSW school wide Cores. The NSCOR Core has the following duties:

1. Interact with Project Leaders and investigators in the design of experiments that use microarray technologies (mRNA, miRNA, DNA copy number, and proteomics) in their experimental approach. Biostatistics and bioinformatics will also be integrated in this process.

2. Establish procedures for consistency in sample preparation and RNA, DNA, and protein extraction, test all RNA, DNA, and protein samples for integrity, and provide reports to that effect.

3. Perform all hybridizations, perform all scans, and place data on a secure server.

4. Provide early analysis on array quality control and preliminary data analysis to investigators.

5. Interact with the Biostatistics and Bioinformatics Core (Core D) to provide bioinformatic analysis of data according to project requirements.

NSCOR Core D: Biostatistics and Bioinformatics. Core Lead: Xian-Jin Xie, Ph.D.; Core Co-Lead: Luc Girard, Ph.D. and Yang Xie, Ph.D. In conjunction with the Genomics (Expression Profiling and Proteomics) Core (Core C) and the Cell Culture Core (Core B), the Biostatistics and Bioinformatics Core (Core D) has the following specific aims:

-To provide database support and expertise for the collection, storage, integration, and retrieval of all sorts of data generated from NSCOR projects.

-To provide valid and optimal statistical design and to conduct proper analysis required to address the specific aims of each project.

-To review background and rationale, to assist in the design, evaluation, and analysis of new research proposal arising from the individual projects.

-To assist the other Cores and Projects in the proper analysis and interpretations of different types of bioassays, particularly mega-data such as those generated from gene expression arrays.

-To assist in manuscript preparation and to review scientific submissions.

-To assist the NSCOR team in building radiation cancer risk models.

As we progress on all the NSCOR projects, significant amount of new data have been generated since our 2007 annual progress report. As a result of Core D effort, a comprehensive data management system has been established that allows efficient access and sharing of all NSCOR data. Data Collection Close to 10,000 samples (including cell pellets and cryogenically preserved cell lines) and experiments (irradiation, microarrays, mouse data) have so far been collected and stored in the NSCOR database. These data can be viewed or edited by any participating investigator provided they have the necessary permissions.

19th Annual NASA Space Radiation Investigators' Workshop, Philadelphia, PA, July 2008. , Jul-2008

EMBO (European Molecular Biology Organization) conference 2008, “Telomeres and the DNA damage response“ ; Villars-sur-Ollon, Switzerland, September 2008. , Sep-2008

Seventh Annual AACR International Conference Frontiers in Cancer Prevention Research, Washington, DC, November 2008. , Nov-2008

Seventh Annual AACR International Conference Frontiers in Cancer Prevention Research, Washington, DC, November 2008. , Nov-2008

44th Annual Meeting of the Radiation Research Society, Boston, MA, September 2008. , Sep-2008

19th Annual NASA Space Radiation Investigators' Workshop, Philadelphia, PA, July 2008. , Jul-2008

8th Annual Hawaii International Conference on Statistics, Mathematics and Related Fields, Honolulu, Hawaii, January 13-15, 2009. , Jan-2009

Eastern North Atlantic Region (ENAR) of the International Biometric Society Workshop, Arlington, VA, March 16, 2008. , Mar-2008

Bayesian Biostatistics Conference, MD Anderson Cancer Center, Houston, Texas, January 2008. , Jan-2008

ICRR meeting, Radiation Research Society, San Francisco, California, July 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators Workshop, Rohnert Park, California, July 13-16, 2007. , Jul-2007

39th Annual Meeting of the EMS (Environmental Mutagen Society), Puerto Rico, October, 2008. , Oct-2008

Specific Aim 1: To quantitate the effects of HZE particle irradiation in causing genetic changes in HBECs and their isogenic derivates.

Specific Aim 2. To quantitate the amount of epigenetic changes caused by HZE particle irradiation in HBECs and their isogenic derivates using high throughput quantitative methylation specific PCR (MSP) assays for acquired promoter methylation

Specific Aim 3. To quantitate the effects of HZE particle irradiation in causing expression profile (mRNA and protein) changes in human bronchial epithelial cells (HBECs) and their isogenic derivates containing various lung cancer-related mutations using array based genome wide and multi protein detecting approaches.

Specific Aim 4. To determine the effect of inter-individual variation on the effects of HZE particle irradiation in causing genetic changes, epigenetic changes, mRNA and protein expression profile changes in human bronchial epithelial cells (HBECs).

We continued studies of gene expression changes following various types of radiation outlined in Aim 3. mRNA gene expression profiles were studied with Illumina v2 arrays before and at different time points after radiation. Supervised clustering methods identified samples by the radiation received via their gene response over time. We also examined kinetic differences rather than static differences as described above. The overall pattern of expression of genes was used to develop predictors of radiation response. From 1480 genes, temporal patterns were developed and 322 identified 12 different patterns

Project 2. Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells.

Aim 1. To test the hypothesis that the immediate cellular response to DNA damage induced by HZE would be different and more complex than that induced by X-rays.

Aim 2. To test the hypothesis that persistent DNA damage induced by HZE is more complex and difficult to repair than that induced by X-rays and, therefore, more deleterious.

Aim 3. To determine if damage induced by HZE is preferentially repaired by non homologous end joining (NHEJ) or homologous recombination (HR) or is preferentially repaired in certain phases of the cell cycle.

Aim 4. To compare the response of human bronchial epithelial cells as non immortalized, immortalized, and with premalignant oncogenic changes in 3D cultures to HZE exposure using a high throughput approach.

Both X-rays and 1 GeV Fe particles elicit similar responses at the sites of DNA breaks. However, Fe-induced damage is repaired more slowly and a large fraction of breaks are not rejoined, thereby resulting in the higher relative biologic effect (RBE) of HZE particles. Using charged particles of increasing molecular weights (O vs. Si vs. Fe) we find that the incidence of un-rejoined breaks increases with increasing particle molecular weight presumably due to increasing complexity of the damage induced. We find that the capacity of both lung fibroblasts and HBECS to repair HZE-induced DNA damage is severely limited compared to gamma rays No major differences are seen between 2D and 3D cultures for fibroblasts. The capacity of HBECs to repair gamma-ray induced DNA damage varies depending upon the cancer-promoting oncogenic changes they harbor – these differences are minimized though in response to 1 GeV Fe.

Project 3. Effect of HZE Particle Irradiation on Functional Progression of Human Lung Cancer

Specific Aim 1. Effects of HZE particles on early events in lung cancer progression (soft agar colony formation and other markers of cellular transformation).

Specific Aim 2. Effects of HZE particles on intermediate events in lung cancer progression (invasion in 3 D organotypic cultures).

Specific Aim 3. Effects of HZE particles on late events in lung cancer progression (ability to form tumors in immune deprived mice).

Soft agar colony forming ability increases for both KRAS+p53 lines as a function of time and dose, and that their response is much greater than for HBEC3-KT (without oncogenic preneoplastic changes). There is no radiation-induced increase in soft agar colony forming ability in cells that over-express either wild type or mutant EGFR. Using quantiative methylation specific PCR (MSP) four of eleven markers found to be methylated are RASSF1A, APC, DcR1, and HCAD with a trend of methylation increasing with time after radiation treatment. We are testing the HZE particle and low LET irradiated HBEC cells of various genotypes for their ability to form tumors in immunodeprived mice as a test of the last events to give full tumor progression. We are also determining the effect of irradiation on the number of cancer stem cell like cells (cancer forming cells) using new sphere forming assays and quantitative mRNA expression profiling for a panel of ~30 stem cell genes.

Project 4. Effects of HZE Particles on the Development of Lung Cancer in Novel Mouse Models

Specific Aim 1. Use K-ras transgenic mouse model of human lung cancer to determine effects of low- and high-LET radiation in both living animals and in isolated tissues.

Throughout the second year of the UTSW NSCOR, experimental groups of approximately one hundred LA1 K-ras animals each were established and irradiated. Two groups were shipped to Brookhaven National Laboratory (BNL) on Long Island, NY for the NSRL-06A and NSRL-06C 56Fe high-LET radiation runs and respectively irradiated with a fractionated or single dose of 1.0 Gy 56Fe particles. Another two experimental groups were irradiated at UTSW with either a fractionated or single dose of 1.0 Gy X-rays for analysis of the effects of low-LET radiation. A fifth group of LA1 K-ras animals was left unirradiated for comparison to all irradiation groups. Irradiation of wild type littermates with a fractionated dose but not a single dose of 1.0 Gy 56Fe particles significantly decreases their survival compared to unirradiated wild-type animals. Mutant animals irradiated with either a fractionated or single dose of 1.0 Gy 56Fe particles have significantly decreased lifespan compared to unirradiated mutant LA1 K-ras mice. In comparison, irradiation with 1.0 Gy X-rays does not affect the survival of mutant or wild type littermate LA1 K-ras mice regardless of administration method. Radiation does not appear to affect overall tumor incidence in irradiated mutant LA1 K-ras mice compared to unirradiated mutant controls. There is a dramatic progression in the grade of lesions compared to unirradiated mutant mice, X-ray irradiated mutant mice, or mutants irradiated with a single 1.0 Gy dose of 56Fe particles. The number adenocarcinomas are significantly increased which display highly aggressive and invasive characteristics of malignancy, which more closely mimics clinical features of lung cancer in humans. Whole genome mRNA analysis was performed and unsupervised cluster analysis of resultant gene expression profiles has suggested divergent responses between mutant and wild type littermate lung associated with radiation type.

Specific Aim 2. Generate or obtain other transgenic mice susceptible to lung cancer to determine the effects of low- and high-LET radiation in both living animals and in isolated tissues. These include the mTERT knockout mouse and the EGFR TK domain mutation mouse. We will initiate pilot experiments of the mTERT KO mouse during the next review period to determine if more end joining, end-end fusions, cytogenetic alterations, micronuclei etc occur when these mice are exposed to 1 and 3 Gy of X-ray. The EGFR mouse model we have obtained has a EGFR mutation that can be induced by tetracycline in the food or drinking water.

Core B. Cell Culture

We placed HBECs into matrigel overlying a lung fibroblast (IMR90) feeder layer and observed that the cells could efficiently differentiate into spheroids or cyst like structures that express SP-A and morphologically resembled small bronchioles and alveolar sacs, and also had lamellar bodies which is a hallmark of type II alveolar cells.

Core C. Genomics and Proteomics Core

This core has started to develop reverse Phase Protein Lysate Arrays (RPPAs). This will allow us to examine the expression of one to several proteins in a moderate throughput platform (hundreds of samples simultaneously). We have also perfected the use of comparative genomic hybridization arrays (aCGH). This will allow us to examine chromosome copy variation which is likely to be important for the analysis of cancer progression studies being carried out in Project 3.

Core D: Biostatistics and Bioinformatics

A centralized database with a server-client architecture has been created to store and share data generated with the various NSCOR experiments. This multi-user database system has already been implemented and extensively tested in other projects and is being adapted and expanded more specifically for NSCOR. The database allows easy access and sharing of all NSCOR data by all investigators. Thousands of collected samples and large sets of data can be organized and shared across investigators and statisticians. Its centralized nature makes it possible to schedule regular backups, to ensure that data are organized in a way that analysis and data interpretation are facilitated, and to allow investigators to keep track of experiments and samples and thereby substantially minimize errors. The database is accessible through the Internet making it possible to enter data in real-time from remote sites such as BNL.

18th NASA investigator’s annual meeting, Monterey , CA, July 13-16, 2007. , Jul-2007

18th NASA investigator’s annual meeting, Monterey/Rohnert Park, CA, July 13-16, 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators Workshop, July, 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators Workshop, July, 2007. , Jul-2007

Warsaw Aging Symposium, Seneca, Warsaw Poland, October 3-7, 2007. , Oct-2007

ISLAC International Congress, Seol Korea, Sept 1-6, 2006. , Sep-2006

Jack Gross Memorial Lecture, Jerusalem, Israel, March 24-28, 2006. , Mar-2006

Beatson International Cancer Conference, Glasgow, Scotland, June 17-20, 2006. , Jun-2006

Distinguished Visiting Professor Pathology Grand Round, Johns Hopkins, November 4, 2006. , Nov-2006

Visiting Professor Lecture, Yale University Medical Center, February 2006. , Feb-2006

Visiting Professor Lecture, Vanderbilt University Medical Center, April 2006. , Apr-2006

Visiting Professor, Lecture, M.D. Anderson Cancer Center, May 2006. , May-2006

16th Annual Space Radiation Health Investigator's Workshop, Port Jefferson, Long Island, NY, May 15-18, 2005. , May-2005

14th International Symposium on Microdosimetry – Venice, Italy, November 13-18, 2005. , Nov-2005

4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators’ Workshop. Moscow – St. Petersburg, June 5 - 9, 2006. , Jun-2006

4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators’ Workshop, Moscow – St. Petersburg, June 5 - 9, 2006. , Jun-2006

4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators’ Workshop, Moscow – St. Petersburg, June 5 - 9, 2006. , Jun-2006

DOE VII Low Dose Program Investigators’ Workshop, Washington, DC, Jan 21-23, 2008. , Jan-2008

NASA Human Research Program Investigators’ Workshop, League City Texas, Feb 3-6, 2008. , Feb-2008

Srinagar, Kashmir India, July 19-25, 2006. , Jul-2006

The overall goals of project 1 are to establish the low LET and the HZE radio-response of human bronchial epithelial cells (HBECs) that have been genetically manipulated, with a variety of oncogenic changes that are often seen during the oncogenic progression towards lung carcinogenesis. These changes include: down regulation of p53; oncogenic KRASV12; and over-expression of wild type and mutant EGFR. These cell lines represent in vitro equivalents of preneoplastic lesions and even with multiple changes, these manipulated HBECs show only part of the malignant phenotype and are not capable of forming tumors in immunodeprived mice. We are performing standard survival analysis following radiation exposure, mRNA gene expression analysis, protein expression, DNA methylation, clonogenicity in soft agar (Project 3), and tumorigenicity (Project 3). We follow irradiated cell cultures up to 4 months post-irradiation in order to examine these endpoints in surviving cells in order to determine the effect the radiation exposure has on the oncogenic process. For low LET radiation exposures the radio-response of these cell lines was not uniform with mutant EGFR containing cells being radiosensitive. Cells exposed to Fe particles at 1000MeV (1 GeV Fe) are highly susceptible to killing, Si particle exposure at the same energy as the Fe particles, produced an intermediate killing effect, while H exposures showed a similar response as that of gamma-radiation. These results correspond well with the ionization intensity differences seen via DNA foci formation in HZE irradiated cells as determined by Project 2. The cells were collected at various time points following irradiation, the RNA was extracted and Illumina expression microarrays were performed. Acute changes (<24 h) were found in gene expression following exposure of human bronchial epithelial cells to irradiation. These changes were distinct for the various types of irradiation (Fe, Si, gamma) as well as for the various HBEC variants. Most experiments showed no long-term gene expression changes as an effect of irradiation.

Methylation of the promoter region of genes is a fundamental process by which tumor cells inactivate tumor suppressor genes. Our studies have focused on the development of methylation following irradiation of human bronchial epithelial cells (HBECs which are devoid of methylation of multiple genes frequently methylated and silenced in lung cancers. We studied the methylation status of 5 genes frequently methylated in lung cancers – RASSF1A, APC, CYT, GRB38 and RAR-beta. Methylation was measured using a TaqMan semi-quantitative real time PCR method. For all of the unmodified HBEC cell groups and for two of the modified HBECKTR53 cell groups, there were no significant differences between radiated and control groups. However for HBECKRASP53 cell group irradiated with HZE particles, there were important differences where the irradiated group which showed methylation of all 5 genes, and a progressive increase in methylation between the 7 and 14 day time points.

Project 2. Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells. David Chen, Ph.D. Project Lead, Sandeep Burma, Ph.D. Co-Lead

With the ultimate objective of estimating cancer risks to humans from HZE particles, in this project we investigate the DNA damage responses of human cells to HZE, the link between these responses and cancer predisposition being very well established. We will take advantage of recent advances in the study of molecular mechanisms of DNA damage sensing, damage signaling, and DNA repair following low LET radiation to verify the hypothesis that HZE particles generate a different and/or unique radiation response in mammalian cells as compared to X-rays. Again we used the panel of isogenic HBEC cells with various oncogenic manipulations described above. Using 1 GeV Fe and X-rays to begin with we have worked out the early damage-responses, damage-repair kinetics, and persistence of DNA damage in primary human skin fibroblasts (HSFs) in great detail. Very briefly, both X-rays and 1 GeV Fe particles elicit similar responses at the sites of DNA breaks. However, Fe-induced damage is repaired more slowly and a large fraction of breaks are not rejoined, thereby resulting in the higher relative biologic effect (RBE) of HZE particles. Using charged particles of increasing molecular weights (O vs. Si vs. Fe) we find that the incidence of unrejoined breaks increases with increasing particle molecular weight presumably due to increasing complexity of the damage induced. Detailed time courses have already been carried out with a panel of HBECs irradiated with 1 GeV Fe particles. The resulting data are currently undergoing analyses.

Project 3. Effect of HZE Particle Irradiation on Functional Progression of Human Lung Cancer. John D. Minna, M.D. Project Lead, Jerry Shay, Ph.D. Co-Lead

Early Events. To evaluate the biologic effects of HZE particle irradiation on HBECs we performed soft agar colony formation assays. Isogenic HBECs with different genetic manipulations were irradiated with HZE and subcultured up to four months after the irradiation. Every month, soft agar colony formation assays were done. The experiments were repeated twice. Most of the oncogenic manipulations reproducibly enhanced the colony formation ability of HBECs without radiation exposure. However, no significant difference was seen between control and HZE-treated HBECs with the one exception of HBECKTR53 (with KRASV12 and p53 knockdown), where HZE irradiated HBECs formed more colonies than non-treated cells at all four time points. This was particularly apparent after correcting for radiation induced decrease in cell survival.

Late Events. We are testing the HZE particle and low LET irradiated HBEC cells of various genotypes for their ability to form tumors in immunodeprived mice as a test of the last events to give full tumor progression. As part of this we are also determining the effect of irradiation on the number of stem cell like cells using new sphere forming assays and quantitative mRNA expression profiling for a panel of ~20 stem cell proliferation program genes.

Project 4. Effects of HZE Particles on the Development of Lung Cancer in Novel Mouse Models. Jerry Shay, Ph.D. Project Lead, James Richardson, DVM, Ph.D.

Transgenic mouse models of human lung cancer have the advantage of a similar tumor development pathway compared to humans but with a shorter developmental period. The LA1 K-ras mouse model we have used contains a latent oncogenic K-ras allele integrated in the endogenous K-ras locus. The activation of the oncogenic allele occurs spontaneously preferentially in the lungs as the animals mature. Every animal containing the latent allele develops alveolar type II lineage lung cancer by the age of four to nine months and all animals die at approximately one year from tumor burden. Our primary objective is to determine if these animals, after low and high LET exposure, initiate or progress to cancer at increased frequency. Our long-term goal is to determine if there is an increase, decrease or no change of onset of death after HZE irradiation and ultimately to use this information for modeling risks in humans. Other end points include other cancer types, DNA damage and repair (gamma-H2AX, DNA-PKc) to correlate with the 2D and 3D cell culture experiments in projects 2 and 3. In addition, we will obtain histology and conduct expression profiles of low and high LET irradiated mouse lungs to compare to the cell culture experiments in Projects 1-3. We have conducted low LET (X-ray) at UT Southwestern Medical Center and high LET (Fe56) at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). 94 animals were irradiated with 0.2 Gy x 5 (1GeV Fe56), 97 animals were irradiated with 1.0 Gy (1GeV Fe56), 110 animals irradiated with 0.2 Gy x 5 (X-ray), and 100 animals irradiated with 1Gy (X-ray). In addition, we have 98 non-irradiated control animals. To examine acute responses (3 hr post fractionated and single dose low and high LET radiation), lung tissue was removed for pathologic examination and RNA isolated for gene expression arrays. Extracted RNA will be hybridized to Sentrix(R) Mouse-6 Expression BeadChips (Illumina). The first section was stained with Hematoxylin and Eosin for histopathological assessment and the following sections are currently being stained with representative markers of the DNA damage response such as gamma-H2AX and 53BP1. Kinetics in normal bronchus, normal alveolus, and tumor tissue will be evaluated separately to determine any differences. We are bringing on line a new mouse model of lung cancer using a transgenic mouse with a Tet-inducible mutant EGFR gene that results in lung cancer. There appears to be a slight decrease in the survival of the irradiated LA1 K-ras mutant and possibly wild-type animals compared to the unirradiated controls. There is also a strain background differences with the B6.1292 background being more susceptible to the radiation effects than the 129sv background. So far, irradiated mutant animals do not appear to have increased tumor incidence as determined by post-mortem evaluation. Overall these experiments allow us to study the effects of HZE and low LET radiation on the development of lung cancer in a whole animal model.

Abstracts, 4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators' Workshop, June 2006. , Jun-2006

Abstracts, 4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators' Workshop, June 2006. , Jun-2006

Abstracts, 4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigators' Workshop, June 2006. , Jun-2006

The overall goals of project 1 are to establish both the low LET and the HZE radio-response of human bronchial epithelial cells (HBECs) that have been genetically manipulated, or not, with a variety of oncogenic changes that are often seen during the oncogenic progression towards lung carcinogenesis. These changes include: the downregulation of p53; the upregulation of oncogenic KRASV12; and the overexpression of wild type and mutant EGFR, as examples. These cell lines represent in vitro equivalents of preneoplastic lesions and even with multiple changes, these manipulated HBECs show only part of the malignant phenotype and are not capable of forming tumors in immunodeprived mice. Characterization of these cell lines includes standard survival analysis following radiation exposure, gene expression analysis, miRNA analysis, protein expression, DNA methylation, clonogenicity in soft agar, and tumorgenicity. Our intent is to follow irradiated cell cultures up to 4 months post-irradiation in order to examine these endpoints in cells that had survived a series of low LET exposures or specific HZE particle exposures in order to determine the effect the radiation exposure has on the oncogenic process. We will also examine inter-individual responses in those endpoints found above that enhance the oncogenic process. Our initial experiments have focused on clonogenic survival after low and high LET radiation exposures. As expected, we found that Fe56 particles are, per unit dose, far more effective at cell killing than gamma-ray exposures. Furthermore, the differences in radiosensitivity seen with low LET exposures between normal cells and cells with mutant EGFR are eliminated after Fe exposure. Si particle exposure, even though at the same energy as the Fe particles produced an intermediate killing effect. This result corresponds well with the ionization intensity differences seen via foci formation in HZE irradiated cells as determined by Project 2. Experiments to determine transformation frequency as a function of radiation exposure type are ongoing (see Project 3). Thus far, no difference has been seen four months after a 1Gy Fe exposure. This dose is highly effective at cell killing and may have eliminated cells that at lower doses would have transformed. Therefore, we are reducing the dose accordingly and re-examining transformation potential. We will continue to fill in the gaps in data for low LET exposure, and now that we have Fe and Si exposed samples, we have started the analysis of the molecular and biochemical endpoints described above, in particular, gene expression analysis. The initial data on gene expression suggest that cells do respond differently to Fe particle exposure as compared to low LET exposures. This response is likely not dose dependent but may reflect an altered kinetic response to the radiation exposure.

Project 2. Effect of HZE particles on DNA damage-sensing and repair pathways in human lung epithelial and fibroblast cells. David Chen, Ph.D. Project Lead, Sandeep Burma, Ph.D. Co-Lead

With the ultimate objective of estimating cancer risks to humans from HZE particles, in this project we investigate the DNA damage responses of human cells to HZE, the link between these responses and cancer predisposition being very well established. We will take advantage of recent advances in the study of molecular mechanisms of DNA damage sensing, damage signaling, and DNA repair following low LET radiation to verify the hypothesis that HZE particles generate a different and/or unique radiation response in mammalian cells as compared to X-rays. As we are specifically interested in risks related to lung cancer development upon exposure to space radiation, we use immortalized normal human bronchial epithelial cell lines (HBECs) and three types of lung fibroblasts (WI38, IMR90 and MRC5) as model systems. Although these HBEC cultures (2D cultures) will be used throughout the project, we will also use normal or premalignant monolayer cultures (2D) and organotypic cultures (3D cultures) to examine these responses in a more in vivo tissue-like setting. The proposed research should reveal fundamental differences in cellular responses between low LET X-rays and HZE exposures in humans and in turn, provide biological markers correlated to lung carcinogenesis for the development of risk assessment and radiation protection for astronauts during future space missions. The objectives of this project are to examine the complexity of DNA damage induced by HZE relative to gamma rays in 2D and 3D cultures of HBECs and to understand the pathways responsible for repair of the complex damage inflicted by these particles and the consequences of inadequate repair responses. Using 1 GeV Fe and X-rays, we have worked out the early damage-responses, damage-repair kinetics, and persistence of DNA damage in primary human skin fibroblasts (HSFs) in great detail. Both X-rays and 1 GeV Fe particles elicit similar responses at the sites of DNA breaks. However, Fe-induced damage is repaired more slowly and a large fraction of breaks are not rejoined, thereby resulting in the higher relative biologic effect (RBE) of HZE particles. Using charged particles of increasing molecular weights (O vs. Si vs. Fe) we find that the incidence of unrejoined breaks increases with increasing molecular weight presumably due to increasing complexity of the damage induced. Detailed time courses have already been carried out with a panel of HBECs irradiated with 1 GeV Fe particles. The resulting data are currently undergoing analyses. Future plans include characterization of these responses in lung fibroblasts and epithelial cells. We have already started analyses of 2D cultures of HBECs that are in different pre-malignant stages of cancer development and in organotypic 3D cultures of lung epithelial cells and fibroblasts.

Project 3. Effect of HZE Particle Irradiation on Functional Progression of Human Lung Cancer. John D. Minna, M.D. Project Lead, Jerry Shay, Ph.D. Co-Lead

This project studies the effect of 2D (tissue culture) and 3D organotypic culture on the response to radiation from high and low LET irradiation. It is important to study the effect of HZE radiation on both the epithelial and stromal (fibroblast) components. Studying the effects of HZE radiation on lung epithelial cells should help elucidate direct mechanisms promoting initial neoplastic transformation as well as progression. Studying the effects of HZE radiation on stromal tissues may uncover other factors contributing to cancer formation and progression such as the disruption of important aspects of cellular cross- talk that promote tissue homeostasis. As described in Project 1, HBECs including HBECs engineered to contain various oncogenic changes were irradiated and then Project 3 tested them for biologic behavior relevant to cancer beginning with studies of anchorage independent growth in soft agar (a characteristic of cancer cells). The HBECs before any changes do not grow in soft agar, but do to varying degrees after these changes. We measured soft agar colony formation 1,2,3, and 4 months after HZE particle irradiation compared to control un irradiated cells. At one month there was increased colony formation in HBECs modified to contain HPV oncogenic E6 and E7 proteins, but not in any of the other 8 HBECs with various oncogenic changes or at other time points. We have also tested the effect of radiation on cells in a 3D (organotypic) system: the fibroblast collagen plug. The lung fibroblast plug closely resembles the stromal pulmonary environment. We irradiated lung fibroblast cells in regular tissue culture and in a collagen plug (3D) arrangement with low and high LET (1 Gy X-ray or 1 Gy HZE Fe56) and studied the DNA damage response and kinetics of repair. After irradiation, the plugs and cellular monolayers were fixed at specific time points (time 0 (control), 2, 12, 24, and 48 hours, and 12 days) and stained using immunofluorescence microscopy for gamma-H2AX as a marker for DNA damage which appears as large discrete foci. Radiation of low and high LET led to damage foci in 90% of cells and the amount of DNA damage was comparable up to 24 hours. However, strikingly, while the low LET radiation DNA damage was resolved by 48 hours, the high LET DNA damage persisted for up to 12 days. We are now testing the 3D cultures with epithelial cells and fibroblasts combined, and we are studying the effects of age, gender, dose rate as well as the gene expression profiles.

Project 4. Effects of HZE Particles on the Development of Lung Cancer in Novel Mouse Models. Jerry Shay, Ph.D. Project Lead, James Richardson, DVM, Ph.D.

Transgenic mouse models of human lung cancer have the advantage of a similar tumor development pathway compared to humans but with a shorter developmental period. The LA1 K-ras mouse model we have used contains a latent oncogenic K-ras allele integrated in the endogenous K-ras locus. The activation of the oncogenic allele occurs spontaneously preferentially in the lungs as the animals mature. Every animal containing the latent allele develops alveolar type II lineage lung cancer by the age of four to nine months and all animals die at approximately one year from tumor burden. Our primary objective is to determine if these animals, after low and high LET exposure, initiate or progress to cancer at increased frequency. Our long-term goal is to determine if there is an increase, decrease or no change of onset of death after HZE irradiation and ultimately to use this information for modeling risks in humans. Other end points include other cancer types, DNA damage and repair (gamma-H2AX, DNA-PKc) to correlate with the 2D and 3D cell culture experiments in projects 2 and 3. In addition, we will obtain histology and conduct expression profiles of low and high LET irradiated mouse lungs to compare to the cell culture experiments in Projects 1-3. We have conducted low LET (X-ray) at UT Southwestern Medical Center and high LET (Fe56) at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). 94 animals were irradiated with 0.2 Gy x 5 (1GeV Fe56), 97 animals were irradiated with 1.0 Gy (1GeV Fe56), 110 animals irradiated with 0.2 Gy x 5 (X-ray), and 100 animals irradiated with 1Gy (X-ray). In addition, we have 98 non-irradiated control animals. To examine acute responses (3 hr post fractionated and single dose low and high LET radiation), lung tissue was removed and RNA isolated for gene expression arrays. We are bringing on line a new mouse model of lung cancer using a transgenic mouse with a Tet-inducible mutant EGFR gene that results in lung cancer. Overall these experiments allow us to study the effects of HZE and low LET radiation on the development of lung cancer in a whole animal model.