FINE MAPPING AND CANDIDATE ANALYSES OF MURINE LUNG TUMOR SUSCEPTIBILITY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Min Wang, B.S.

*****

The Ohio State University

2003

Dissertation Committee: Approved by

Professor Ming You, Advisor Advisor Professor Christoph Plass, Co-advisor

Professor Gary Stoner Co-advisor Molecular Virology, Immunology and Professor Yian Wang Medical Genetics Graduate Program

ABSTRACT

Lung cancer is the leading cause of cancer death in men and women in the United

States. While the exposure to tobacco smoking and other environmental carcinogens represents the main risk factors, it has been clear that inherited components may be also involved in the development of lung cancer. Segregation analysis has revealed that susceptibility of the human population to different forms of lung cancer follows a pattern of autosomal dominant Mendelian inheritance. However, due to the genetic heterogeneity, substantial variance in exposure to environmental risk factors, poor prognosis and other factors, identification of human lung cancer susceptibility genes by direct linkage analysis is difficult. Mouse inbred strains show widely different susceptibilities to both spontaneously occurring and chemically induced lung tumorigenesis and become a valuable model to study lung cancer genetics. To date, linkage analyses using various mouse crossing panels have uncovered more than 20 putative lung tumor susceptibility /resistance loci on mouse . Considering the highly homologous relationship between mouse and human genomes, identifications of these putative loci may ultimately lead to the discovery of human lung cancer susceptibility genes.

A quantitative trait (QTL), Pulmonary adenoma susceptibility 1 (Pas1), was previously mapped to the distal region of mouse 6 using genetic linkage ii analyses on crosses between susceptible A/J and resistant C3H/He or C57BL/6J strains.

This locus accounts for approximately 40-60% variance in lung tumor multiplicity induced by chemical carcinogens between the A/J and C3H/He or C57BL/6J mice. In addition to predisposing lung tumor multiplicity, the Pas1 locus also affects mouse lung tumor sizes. Although it is the major QTL in predisposition to mouse lung tumor susceptibility, the Pas1 gene(s) has not been identified.

In the present studies, we have fine-mapped the Pas1 locus using two independent strategies. We have utilized a newly developed F11 generation of Advanced Intercross

Line (AIL) mouse population to fine map Pas1, Pas2 (on chromosome 17) and Pas3 (on chromosome 19) QTLs. By selectively genotyping 30% of the population, we have confirmed the Pas1 QTL and refined it into an interval of approximately 1.0-cM (~1.3

Mb) in the vicinity of the Kras2 gene. The Pas2 QTL was detected by both ANOVA and regression analysis but not by Mapmaker software. An interaction between the Pas1 and

Pas2 QTLs was also revealed. However, the Pas3 QTL has not been confirmed in this study.

Congenic strategy was also utilized to fine map the Pas1 QTL. Like in AIL project, we started with two parental strains: lung tumor susceptible A/J strain and lung tumor resistant C57BL/6J strain. After seven generations of backcrossing, N7 congenic mice that carried ~36Mb of the A/J Pas1 QTL region were further crossed to the

C57BL/6J mice to generate subcongenic strains of mice (N8), which contain various

iii Pas1 QTL regions. N9 mice, generated by mating each selected N8 male mouse with

three C57BL/6J female mice, were treated with an initiating dosage of carcinogens and

were allowed to form lung tumors. A set of new polymorphic markers has been

developed to assist this fine-structure mapping. Combining results from the AIL project

and the congenic project, we have refined the Pas1 QTL into a less than 1-Mb minimum

candidate region based on the Celera/or public mouse genome maps encompassed by the markers D6Osu6 and D6Osu11.

Based on the fine-mapping results, the Pas1 locus was then sufficiently fine- mapped that candidate gene screening for the Pas1 locus could be performed. After initial screening, six genes located in the minimum Pas1 candidate region were selected for further examinations. The Lrmp/Jaw1 gene bears amino acid polymorphisms tightly co- segregating with strain Pas1 alleles. The RIKEN Ak016641 (re-named as Pas1c1 for

Pas1 candidate 1) gene, encoding an intermediate filament tail domain-containing , produces alternatively spliced transcripts in inbred strains of mice and also Mus

Spretus. Its mRNA splicing pattern tightly co-segregates with Pas1 alleles. Another novel gene, Pas1c2, identified in our study, was found bearing an amino-acid changing nucleotide polymorphism between lung tumor susceptible and resistant strains, which is also tightly correlated with strain Pas1 status. Thus, our results support these three genes as strong candidates for the Pas1 QTL, and they are being further tested by in vitro and in vivo functional analyses. We also examined the other three genes (Eca39, RIKEN

iv Ak015530 and mHoj-1). Neither functional polymorphism nor expression difference has been found for Eca39 and mHoj-1 genes between lung tumor susceptible and resistant

strains. The Ak015530 gene carries an amino-acid polymorphism but this polymorphism

does not co-segregate with mouse lung tumor susceptibility. Thus, these three genes are

less likely candidates for the Pas1 locus.

Genetic studies have mapped a lung tumor resistance locus designated as Par2 to mouse . Par2 accounts for approximately 40% of the difference in lung tumor multiplicity between the susceptible A/J mice and the relative resistant BALB/c mice. We have fine mapped the Par2 locus using congenic mice that were constructed by

placing an approximately 28-cM Par2 QTL region from the A/J mice onto the genetic

background of BALB/c mice. Subcongenic strains of mice (N8) containing various Par2

QTL regions were generated and genotyped. Like in Pas1 congenic project, the N9 mice were generated by mating each selected N8 male mouse with three BALB/c female mice and were treated with urethane to induce lung tumor formation. Consequently, by analyzing the lung tumor multiplicity and genotypes of each subcongenic strain, the Par2 locus was narrowed to an approximately 6.3-Mb region flanked by the marker

D18Mit103 and the marker D18Mit162. A mouse/human comparative map was constructed for the Par2 candidate region based on Celera genomic information. A high homology between the mouse Par2 candidate region and the human syntenic region on chromosome 18q21 has been observed. Based on recent mouse genome maps, there are

v totally 79 putative genes residing in the candidate region. A subset of these genes was found to exhibit differential in lungs between the A/J and BALB/c mice when assayed by real-time RT-PCR. Four genes are possible Par2 candidates based on this assay, including Myo5b, Smad7, Mapk4, and GABA-A receptor-like gene mCG58197.

More interestingly, sequencing analyses for this region found that the Rad30b gene, encoding the DNA–dependent polymerase iota (Polι), carries 25 nucleotide polymorphisms in its coding region between A/J and BALB/c inbred strains, causing ten amino-acid alterations. Functional analyses on the above five genes are needed to clarify their Par2 candidacy. In addition, 54 putative genes exhibited various types of SNPs when comparing genomic sequences from A/J, 129X1/svJ, 129S1/svImJ, DBA/2J, and

C57BL/6J mice. Screening of these SNPs in the future may help us find polymorphic markers and identify new Par2 candidate genes.

vi

Dedicated to my family, my mentors and all my friends

vii ACKNOWLEDGMENTS

I wish to thank very much my advisor, Dr. Ming You, for his intellectual

support, encouragement, and enthusiasm that made this thesis possible, and for his

patience in correcting both my stylistic and scientific errors.

I wish to thank my other advisory committee members, Dr. Christoph Plass, Dr.

Gary Stoner and Dr. Yian Wang, for their continual devotion of precious time and energy

to my Ph.D graduate training.

I would like to thank our collaborators: Dr. Alvin M. Malkinson from University

of Colorado, Dr. Fuad Iraqi from International Livestock Research Institute in Kenya and

Theodora R. Devereux from National Institute of Environmental Health Science, for their

intellectual and material supports to our projects.

I am grateful to Dr. Zhongqiu Zhang, Dr. William J. Lemon and Dr. Futamura

Manabu for their critical contribution to the present projects and intellectual discussion

during my research.

I appreciate the opportunity of working in such a wonderful laboratory. I thank my colleagues in this laboratory for all the helps I have received from them during my research.

Finally, I cannot thank enough to my family for their both mental and physical support during my Ph.D. training period. viii VITA

Dec.12, 1975………………………..Born, Hubei Province, P. R. China

1997…………………………………BS in Biology, South China Normal University, P. R. China

1997-1998……………….………….MS student, Dept. of Cancer Biochemistry, Guangzhou Medical College, P. R. China

1998-1999…………………………..MS student, Dept. of Pathology, Medical College of Ohio, Toledo, Ohio

1999-2000…………………………..Ph.D. fellowship, Molecular Basis of Disease Program, Medical College of Ohio, Toledo, Ohio

2000-present………………………...Ph.D. Graduate Research Assistant, Molecular Virology, Immunology and Medical Genetics Program, The Ohio State University, Ohio

ix PUBLICATIONS

Research Publication

1. Fine mapping of Pulmonary adenoma susceptibility1 (Pas1) gene using congenic mice. (Temporary title and in preparation)

2. Identification of Pulmonary adenoma resistance 2 (Par2) gene(s) on mouse chromosome 18. (Temporary title and in preparation)

3. Wang M, Lemon WJ, Liu GJ, Wang Y, Teale AJ, Iraqi F, Malkinson AM, and You M. Fine mapping of the pulmonary adenoma susceptibility gene 1 (Pas1) locus using advanced intercross lines. Cancer Research 2003 Jun 15, 63 (12)

4. Lemon WJ, Swinton CH, Wang M, Berbari N, Wang Y and You M. SNP analysis of mouse pulmonary adenoma susceptibility loci 1-4 for identification of candidate genes. J Med Genetics 2003 Apr; 40(4): e36

5. Ozbun LL, Martinez A, Angdisen J, Umphress S, Kang Y, Wang M, You M, Jakowlew SB Differentially expressed nucleolar TGF-beta1 target (DENTT) in mouse development. Dev Dyn 2003 Mar; 226(3): 491-511

6. Wang Y, Hu L, Yao R, Wang M, Crist KA, Grubbs CJ, Johanning GL, Lubet RA, You M. Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor. Oncogene 2001 Nov 22; 20(53): 7710-21

7. Zhang Z, Lin L, Liu G, Wang M, Hill J, Wang Y, You M, Devereux TR. Fine mapping and characterization of candidate lung tumor resistance genes for the Par2 locus on mouse chromosome 18. Exp Lung Res 2000 Dec; 26(8): 627-39

FIELDS OF STUDY

Major Field: Molecular Virology, Immunology and Medical Genetics

x TABLE OF CONTENTS

Page

Abstract…………………………………………………………………………… ii

Dedication…………………………………………………………………………. vii

Acknowledgments……………………………………………………………….… viii

Vita………………………………………………………………………………… ix

List of Tables……………………………………………………………………... xv

List of Figures………………………………………………………………..…… xvi

Chapters:

1. Introduction………………………………………………………………... 1

1.1 Human lung cancer ……………………………………………………. 1

1.1.1 Incidence, risk factors and mortality rate…………………... 1

1.1.2 Diagnosis and treatment……………………………………… 2

1.1.3 Molecular genetics of human lung cancer…………………… 4

1.1.3.1 RAS oncogene activation……………………………… 4

1.1.3.2 MYC family genes……………………………………. 6

1.1.3.3 p53 …………………………. 6

1.1.3.4 p16INK4a /Cyclin D1/CDK4/ RB pathyway…….……… 7

1.1.3.5 Short arm of deletion and other

chromosomal abnormalities………………………... 9

xi 1.1.3.6 ERBB-2 receptor tyrosine ……………..…… 10

1.1.3.7 …………………………………………... 11

1.1.3.8 Others………………………………………………… 12

1.2 Genetic inheritance in human lung cancer…………………………… 12

1.2.1 Familial aggregations of human lung cancer ………………. 13

1.2.2 Human lung cancer susceptibility candidate genes ………. 15

1.2.3 Challenges in identification of human lung cancer

susceptibility genes………………………………………….. 16

1.3 Genetic dissection of lung tumor susceptibility using inbred

strains of mice…………………………………………………………. 17

1.3.1 Similarity between human adenocarcinomas (AC) and mouse

lung tumors…………………………………………………… 18

1.3.1.1 Similarity in anatomical origin of tumors………….. 18

1.3.1.2 Similarity in molecular characteristics…………….. 19

1.3.2 Inbred strains of mice show different lung tumor

susceptibility …………………………………………………. 20

1.3.3 Mouse lung tumor susceptibility governed by Pulmonary

adenoma susceptibility/resistance genes…………………… 20

1.4 Positional cloning of lung tumor susceptibility/resistance genes…… 23

1.4.1 Fine-mapping strategies for mouse gene cloning…………… 25

xii 1.4.2 Fine-mapping strategies used in present studies…………… 26

1.4.2.1 Congenic strategy…………………………………….. 26

1.4.2.2 Advanced Intercross Line (AIL) mouse

population…………………………………………….. 27

1.4.3 Mouse genome projects and gene cloning…………………… 27

1.5 Objective and summary of the study………………………………… 29

2. Fine mapping of Pulmonary adenoma susceptibility 1,2,3 (Pas1, 2, 3)

using Advanced Intercross Line (AIL)mouse population………………. 34

2.1 Introduction…………………………………………………………….. 34

2.2 Materials and methods………………………………………………… 37

2.3 Results………………………………………………………………….. 40

2.4 Discussion………………………………………………………………. 42

3. Fine mapping of Pulmonary adenoma susceptibility 1 (Pas1) QTL using

congenic mice……………………………………………………………….. 50

3.1 Introduction……………………………………………………………… 50

3.2 Materials and methods…………………………………………………. 52

3.3 Results…………………………………………………………………… 56

3.4 Discussion……………………………………………………………….. 59

xiii

4. Candidate gene screening for Pulmonary adenoma susceptibility 1 (Pas1)

QTL………………………………………………………………………….. 69

4.1 Introduction……………………………………………………………… 69

4.2 Materials and Methods………………………………………………… 70

4.3 Results………………………………………………………………….. 74

4.4 Discussion………………………………………………………………. 80

5. Fine mapping and candidate gene analyses for mouse Pulmonary

adenoma resistance 2 (Par2) QTL……………………………………….… 95

5.1 Introduction……………………………………………………………. 95

5.2 Materials and methods………………………………………………… 99

5.3 Results………………………………………………………………….. 104

5.4 Discussion…………………………………………………………….… 110

6. Future directions…………………………………………………………… 125

Bibliography…………………………………………………………………….. 129

xiv LIST OF TABLES

Table Page

1.1 Variant pulmonary adenoma susceptibility in inbred strains of mice… 32

1.2 Pulmonary adenoma susceptibility (Pas) loci in mouse genome ……… 33

1.3 Pulmonary adenoma resistance (Par) loci in mouse genome………….. 33

2.1 ANOVA analyses on Pas QTLs …………………………………………. 49

3.1 Pas1 QTL effect on both lung tumor multiplicity and size……………. 66

3.2 New polymorphic markers between D6Mit59 and D6Mit15 …………. 67

3.3 Genes located in the minimum Pas1 candidate region ………………… 68

4.1 Comparison of the mouse genome maps from the Celera Genomics

and the Mouse Genome Sequencing Consortium(MGSC)……………. 84

4.2 AA-changing nucleotide polymorphisms in the Pas1c2, Lrmp,

Ak015530 and Pas1c1 (Ak016641) genes………………………………. 91

5.1 Real-time RT-PCR gene expression analyses in the Par2 region

reveal some genes differentially expressed in A/J and BALB/c

mouse lung tissue………………………………………………………… 121

5.2 Amino-acid polymorphisms between A/J and BALB/c Rad30b genes 122

5.3 Celera computer-predicted Single Nucleotide Polymorphisms (SNPs)

in the Par2 region………………………………………………………… 123

xv

LIST OF FIGURES

Figure Page

2.1 MapmakerEXP/QTL analysis on Pas loci……………………………… 45

2.2 Linear multiple regression analysis of Pas loci…………………………. 47

3.1 Flowchart of fine mapping Pas1 locus using congenic strategy………. 63

3.2 Genetic linkage map and the Pas1 QTL on mouse ……. 64

3.3 Fine mapping of Pas1 QTL using congenic mice……………………… 65

4.1 Pas1c2 gene Open Reading Frame (ORF)………………………………. 85

4.2 Pas1c2 protein homology among mouse, human and Ciona intestinalis 87

4.3 Nucleotide polymorphisms in the Lrmp gene that alter amino acids …. 89

4.4 RT-PCR expression pattern of six genes……………………………….. 92

4.5 Alternative splicing of Ak016641 mRNA………………………………… 94

5.1 Tumor multiplicity of congenic strain AC and control strain CC……. 116

5.2 Congenic mapping of the Par2 QTL……………………………………… 117

5.3 Mouse / Human comparative map for the Par2 candidate region……… 119

xvi CHAPTER 1

INTRODUCTION

1.1 Human lung cancer

1.1.1 Incidence, risk factors and mortality rate

According to the statistics of American Cancer Society, about 1,334,100 new

cancer diagnoses are expected in the United States in 2003 and about 556,500 Americans

are expected to die of cancer(1). Among all types of cancers, lung cancer is the leading

cause of cancer death. An estimated 171,900 new lung cancer cases are expected in 2003,

accounting for about 13% of new cancer diagnoses. About 157,200 Americans are

expected to die from lung cancer this year, which accounts for 29% of all cancer deaths.

Tobacco smoking is the most important cause of lung cancers with 80%~90% lung cancer cases arising in cigarette smokers (2). A lifetime smoker has a ~13 fold increased risk of developing lung cancer compared to a lifetime nonsmoker(3). There is also mounting evidence for an increased risk of lung cancer from exposure to environmental tobacco smoke (ETS). Epidemiological, biochemical, and toxicological

1 studies indicate there is a causal association between ETS and lung cancer. In a large

meta-analysis of 4,626 cases the excess risk of lung cancer was 24% (95% confidence

interval 13% to 36%) in non-smokers who lived with a smoker(4). Other risk factors

include occupational or environmental exposures to substances such as arsenic, radon,

asbestos, mustard gas, polycyclic hydrocarbons, chloromethyl ethers, chromium, etc (5-

7).

Despite advances in diagnosis and treatment of the disease made in the past two

decades, the prognosis for lung cancer patients remains dismal with an overall 5-year

survival rate of less than 15% (8). This high mortality rate is primarily due to the lack of

effective methods for early detection and strategies for treating patients with advanced

stages of lung cancer.

1.1.2 Diagnosis and treatment

For treatment purposes lung cancer is divided into two histological classes, small cell lung cancer (SCLCs) and non-small cell lung cancer (NSCLCs), which differ in their responses to therapy. Approximately 80% of lung cancer cases are classified as NSCLCs, while SCLCs account for ~18% (9). NSCLCs can be histologically further divided into three major subtypes: squamous cell carcinoma (30%), adenocarcinoma (40%) and large cell carcinoma (15%)(10).

2 The evaluation of the lung cancer patients involves obtaining material for a

histologic diagnosis, determining the stage of the disease, computed tomography (CT) scans of the chest and abdomen, pulmonary function tests, routine laboratory test and weight loss of patients. Direct examination of large airways and obtaining biopsy material is often performed using flexible fiberoptic bronchoscopy (11). Improved bronchoscopy such as fluorescence bronchoscopy is also often used to reveal small or subtle cancerous and precancerous bronchial lesions (12). In addition, a more advanced

18F-2-fluoro-2-deocyglucose (FDG) positron emmission tomography (PET) scan has been

introduced to improve staging (13).

Treatment options are based on the lung cancer stage at the time of diagnosis. It is

common that SCLC tumors have metastasized to distant organs by the time of diagnosis.

Due to its propensity for metastasis, SCLC patients are rarely treated by surgical

resection. Patients with chest-restricted SCLCs often receive concurrent radiotherapy and

chemotherapy. Combination chemotherapy regimens that include a platinum agent

(cisplatin or etoposide) and other anti-lung cancer agents such as paclitaxel are the

standard of care for most “extensive-stage” SCLC patients (11). However, although most

SCLCs are initially highly responsive to therapy, typically the primary tumor or

metastasis will finally become resistant to chemotherapy and vast majority of patients succumb to the disease. Most of NSCLC patients are treated with surgery to remove the primary tumor, followed by chemotherapy and/or radiation. For patients with

3 unresectable metastatic disease, platinum compounds-based combination chemotherapy

is again the treatment of choice. However, just like patients with SCLCs, most of such

NSCLC patients become resistant to therapy, relapse and die from the disease.

1.1.3 Molecular genetics of human lung cancer

Like many other cancers, lung cancer development is a multistep process where

genetic alterations accumulate to cause the disease. Advances in the understanding of the

molecular events underlying the development of lung cancer may enable researchers to

develop rationally targeted therapies providing treatment options with increased efficacy

and decreased toxicity.

Over the past two decades, significant progresses have been made toward understanding the molecular pathogenesis of human lung cancer by the identification and

characterization of various cancer-related genes that are genetically and/or epigenetically

altered in human lung cancers (14-20).

1.1.3.1 RAS oncogene activation

The RAS gene family (K-RAS, H-RAS and N-RAS) encodes similar 21 KD membrane-bound and is activated by point mutations at codons 12,13 or 61 in approximately 20-30% lung adenocarcinomas and 15-20% of all NSCLCs, but very rarely in SCLCs (14). Activating mutations in the K-RAS proto-oncogene are found in

4 about 90% of RAS mutations in lung adenocarcinomas. Point mutations at codon 12 are

the most frequent with 85% of K-RAS mutations affecting codon 12, followed by

mutations at codons 13 and 61.

Mutations at these GTPase-sensitive sites on the K-RAS protein hampered the

hydrolysis of GTP to GDP, which prolongs the activation of K-RAS and the downstream

signaling pathways. Once activated, K-RAS stimulates multiple downstream

effectors(21). K-RAS activation of the RAF serine/threonine and the subsequent

activation of the ERK mitogen-activated protein kinases (MAPKs) remains a key signal

transduction pathway. The second best- characterized K-RAS effector is

phosphatidylinositol 3-kinases (PI3 kinases), which function in converting

phosphatidylinositol (4,5)-bisphosphate [PtdIns (4, 5) P2] to phosphatidylinositol (3,4,5)-

trisphosphate [PtdIns (3,4,5) P3]. Increased PtdIns (3,4,5) P3 in turn upregulated

AKT/PKB and RHO family protein RacGTPase activity, which may promote cell

survival and suppress apoptosis (22-26). The third characterized class of K-RAS effectors is GDP-GTP exchange factors (GEFs) for the RAL small GTPase (RalGDS, RGL, RGL3 and RGL2/Rif). Although found to not be sufficient to transform rodent fibroblasts, the activation of RalGEF effector pathway may be critical for K-RAS transformation in human cells (27).

5 1.1.3.2 MYC family genes

MYC proto-oncogene family genes (C-MYC, N-MYC and L-MYC) encode for cycle specific nuclear phosphoproteins which function as basic helix-loop-helix leucine- zipper (bHLHZ) class of factors and are involved in the regulation of expression of other cell growth, differentiation and apoptosis-related genes. In contrast to

RAS oncogenes, point mutations of MYC family genes are rare in human lung cancers.

Activation of the MYC family genes has been observed mainly by gene amplification or transcriptional dysregulation, with C-MYC gene being the most frequently activated in lung cancer, particularly in SCLCs (28). Amplification and overexpression of MYC family genes appear to be more prevalent in tumor cell lines than in tumor tissues, and in

SCLCs than in NSCLCs (19). Results of numerous studies of MYC gene amplification have shown that 18% of SCLC tumors and 31% of SCLC cell lines had gene amplification of one member of the MYC family. In terms of NSCLC, 8% of NSCLC tumors and 20% of NSCLC cell lines had gene amplification of one member of the MYC family (19).

1.1.3.3 p53 tumor suppressor gene

The p53 tumor suppressor gene is located at chromosome 17p13 and encodes a

“Gatekeeper” nuclear protein involved in cell cycle control, DNA repair, cell differentiation, genomic instability, programmed cell deaths and cell senescence (29, 30).

6 Activated by DNA damage signals, hypoxia, or inappropriate cell growth signals, such as

those derived from ectopic expression of oncogenic K-ras gene or dysregulated E2F

protein level due to the loss of Rb tumor suppressor gene function, the p53protein

functions as a transcription factor to regulate expression of a broad range of downstream

genes including p21/WAF1/CIP1, MDM2 and BAX etc, leading to transient cell cycle

arrest and/or apoptosis.

Mutation of the p53 gene is the main event that leads to the inactivation of the p53 stress-response pathway in lung cancers (31). The p53 mutations are among the most frequent abnormalities occurring in 75-100% of SCLCs and ~50% of NSCLCs (32).

Most mutations are missense mutations occurring in the mid region of the gene (exon 5-

8), leading to both gain of new function and loss of wide type p53 function. Due to its short half-life, the wild type p53 protein is undetectable by immunohistochemistry under normal physiological conditions. However, the p53 mutations prolong the half-life of the p53 protein to several hours leading to increased protein levels readily detected by immunohistochemistry. p53 mutations have been detected in preneoplastic lesions of the lung, suggesting that they occur early during lung carcinogenesis (33, 34).

1.1.3.4 p16INK4a /Cyclin D1/CDK4/ RB pathyway

The retinoblastoma gene (RB) is located in human chromosomal region 13q14, encoding a nuclear phosphoprotein that was initially identified as a tumor suppressor

7 gene in childhood retinoblastomas. The hypophosphorylated RB protein acts as a growth

suppressor by inactivating a number of proteins including the transcription factor E2F1,

which promote transcription of genes required for DNA replication, thus blocking the

G1/S transition. RB mutations are found in 90% of SCLCs and 15-30% of NSCLCs (35-

37). Most mutations result in truncated proteins (38).

The p16INK4a gene is located on chromosome 9q21 and encodes a cell cycle

inhibitor that prevents RB protein hyperphosphorylation by inhibiting Cyclin D1/CDK4

kinase activity. While RB inactivation is the preferential mechanism in SCLCs, p16INK4a abnormalities are found frequently in NSCLCs. Inactivation of p16INK4a is caused by a

variety of mechanisms including mutations, deletions or promoter hypermethylation. The

rate of mutation of p16INK4a is relatively low in NSCLCs while homozygous deletions have been observed in 10-40% of the tumors (39, 40). Hypermethylation in the 5’-CpG island of the p16INK4a gene was suggested to cause the downregulation of p16INK4a in lung

cancer carrying no genetic mutations of p16INK4a (41, 42). Taken together, the inactivation

rate of p16INK4a is 30-70% in NSCLCs (14).

CyclinD1/Cyclin Dependent Kinase 4(CDK4) complex phosphorylates RB and

inactivates RB binding activity to the transcription factors essential for the G1/S entry

(e.g. E2F1 protein). Overexpression of Cyclin D1 is found in majority of NSCLC cell lines as a result of abnormal gene amplification. In primary NSCLC tumors, 12-40 % of

NSCLC tumors overexpress CyclinD1 through gene amplification or other mechanisms

8 (43, 44). The role of CDK4 in lung cancer is unknown, but it has been reported that

CDK4 is expressed in about 90% of NSCLCs and the expression associated with poor differentiation (45).

1.1.3.5 Short arm of chromosome 3 deletion and other chromosomal abnormalities

Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer, and is observed in more than 90% of

SCLCs and in 50–80% of NSCLCs (46, 47). At least three distinct 3p regions---3p14-cen,

3p21.3, 3p25-26--- have been identified by alleotyping, suggesting that there are probably over three different tumor suppressor genes located on chromosome 3p.Many candidate genes have been identified in these regions, including FHIT gene at 3p14.2 and RASSF1A gene at 3p21.3.

The FHIT (Fragile Histidine Triad) gene encodes a 16.8 KD dinucleoside 5′, 5′′′-

P1, P3- triphosphate (Ap3A) hydrolase (48). It encompasses approximately 1 Mb of

genomic DNA which includes the human common fragile site (FRA3B). Common fragile sites have been shown to display a number of characteristics of unstable, recombination- prone DNA and are preferential sites for chromosomal deletion and rearrangement (49).

Small cell lung tumors (80%) and non-small cell lung tumors (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles (50). Absence of FHIT protein in some precancerous dysplastic lesions has been

9 observed suggesting that FHIT inactivation may occur at an early phase of lung carcinogenesis (51).

RASSF1A (Ras ASSociation domain Family 1A) gene encodes a 38.8-kD protein containing a RAS association (RalGDS/AF-6) domain (RA). It was found by Pfeifer’s group that the expression of RASSF1A was epigenetically inactivated in 40% of primary

NSCLCs and 100% (17 of 17) of analysed SCLC cell lines through hypermethylation of the CpG island promoter sequence (52). A later study from the same group has shown in

22 of 28 (=79%) SCLC tumors the promoter of RASSF1A was highly methylated at all

CpG sites analysed (53). John Minna’s group also published results showing RASSF1A promoter hypermethylation in 100% of SCLC tumors and 63% of NSCLCs (54). The role of RASSF1A as a tumor suppressor gene has been reinforced by the observation that re- expression of RASSF1A in A549 lung cancer cells reduced colony formation, suppressed anchorage-independent growth and inhibited tumor formation in nude mice (52).

In addition to 3p, it has been reported that chromosomal gains are frequent in 5p,

8q, 17q and 19q in NSCLCs and 3q, 5p, 8p and Xq in SCLCs, whereas chromosome losses are frequent in 1p, 4q, 5q, 6q, 8p, 9p, 13q and 17p in NSCLC and 5q, 13q and 17p in SCLC (55-59).

1.1.3.6 ERBB-2 receptor tyrosine kinase

The ERBB-2 (HER/neu) oncogene, which has been mapped to chromosome

10 17q21, encodes for a transmembrane receptor protein (p185) with tyrosion kinase

activity. Activation of ERBB-2 occurs by gene amplification and/or overexpression and

can be detected in several types of human tumors (60, 61), especially in breast and

ovarian cancers. In lung cancers, ERBB-2 gene is highly expressed in over a third of

NSCLCs, especially adenocarcinomas (62-64). On the other hand, ERBB-2

overexpression has been also correlated to the lung tumor responsiveness to

chemotherapy, especially in NSCLCs (65).

1.1.3.7 Telomerase

Telomerase is not expressed in normal somatic tissues but highly expressed in

over 90% of human tumor cell lines and tumor tissues (66). Recent studies have revealed

that activation of telomerase is an essential step to bypass replicative senescence and to

allow for transformation of human cells (67). Analysis of lung samples showed that

telomerase activation was observed in 100% of SCLCs and 85% of NSCLCs (68, 69). In

addition, reactivation of telomerase activity may be an early event in lung tumorigenesis

(70). Since the is not present in normal tissues (except germ cells), telomerase may become a good target for therapeutic intervention.

11 1.1.3.8 Others

PTEN/MMAC1 gene located at 10q23 was identified as a downstream target for

transforming growth factor-beta (TGF-β). It has been reported that approximately 30% of

SCLCs and 5% of NSCLCs contain inactivating mutations (71-74). Hemizygous loss of

PTEN is also detected in 50% of NSCLCs which may affect gene function through gene

dosage alteration (haploinsufficiency) (75-77).Over expression of epidermal growth

factor receptor (EGFR) and its ligand TGF-α has been found in about 60% of NSCLCs

(78-80). Expression of neuroendocrine factors, including gastrin-releasing

peptide/bombesin-like peptides (GRP/BN), and their receptors, are widely expressed in

SCLCs. Their expression can be detected in almost all SCLC cell lines and one half of

the primary SCLCs (19). Overexpression of the anti-apoptosis BCL-2 gene have been

observed in 80% of SCLCs and 10-30% of NSCLCs (81-83). SMAD2 and SMAD4 at the

chromosome18q21 have shown low frequency of somatic missense and frameship

mutations in lung cancer (84, 85).

1.2 Genetic inheritance in human lung cancer

It is widely acknowledged that cigarette smoking is a dominant risk factor for lung cancer, and that carcinogens in the smoke are main initiators of lung cancer by

12 inducing multiple genetic alterations, mainly through the formation of DNA adducts.

However, although cigarette smoke is implicated in about 90% of male and 75-80% of female lung cancer deaths (86), only about 10% of heavy smokers will ultimately develop lung cancer (87). This suggests that there is a variation in individual susceptibility to lung cancer.

1.2.1 Familial aggregations of human lung cancer

In 1963, Tokuhata and Lilienfeld provided the first epidemiologic evidence for

increased familial risk for lung cancer (88). Analyses of data from a comparison of

relatives of 270 lung cancer probands with those of the race-sex-age-residence matched

controls indicated significant excess in the lung cancer mortality among proband

relatives. While the role of cigarette smoking in lung cancer development was confirmed

in the study, their further detailed analyses also revealed that lung cancer aggregated in

families with or without the influence of cigarette smoking and the degree of such

aggregation is more marked among nonsmoker than among smokers (88). Their result

has demonstrated a synergistic interaction between the genetic factor and cigarette

smoking to produce a “booster effect” on developing lung cancer.

Since then, lung cancer familiar aggregation has been confirmed in several other

studies. By describing 58-year-old identical twin men, Joishy et al. suggested that a

gene(s) shared by these twins not only determines the susceptibility of pulmonary cells to

13 malignant transformation by oncogenic agents but also affects the histological features and metastatic behavior of the resulting neoplasm (89).

Ooi et al reported an increased familiar risk (2.4-fold greater) for lung cancer among relatives of 337 lung cancer probands after allowing for age, sex, smoking and occupational-industrial exposure (90). Taking into account of lung cancer histological type, Lynch et al. have reported the finding of heterogeneity of risk among patient families with an increased risk only among relatives of patients with lung adenocarcinomas (91). McDuffie investigated familial, generational and site clustering of cancer in families of patients with primary lung cancer. His result indicated that patient families were more likely to have two or more affected members, a multigenerational pattern and a greater tendency for documented multiple primary tumors in the same individual, suggesting that a positive family history of lung cancer is a risk factor which interacts with environmental exposure (92).

Shaw et al. evaluated lung cancer cases/controls in Texas and reported that compared with 9% of controls, 14.5% of lung cancer cases had at least one first degree relative with lung cancer. Furthermore, individuals with squamous cell carcinoma or adenocarcinoma were more likely to have a family history of cancer than individuals with small cell carcinoma (93). A recent study conducted by Schwartz et al. revealed that a first-degree relative was associated with a 7.2-fold increased lung cancer risk among non- smokers in the 40- to 59-year-old age group. The increased risk remained significant

14 (relative risk=6.1) after adjustment for the smoking, occupational and medical history of each family member (94).

1.2.2 Human lung cancer susceptibility candidate genes

In 1990, Seller et al. published results of segregation analyses on 337 families of lung cancer probands. After allowance for variable age of onset of lung cancer and smoking history, the results indicated susceptibility to lung cancer may be inherited in a

Mendelian co-dominant fashion and suggested the existence of a rare major autosomal cancer gene that produced earlier age of onset of lung cancer (95). Segregation at this putative locus could account for 69% and 47% of the cumulative incidence of lung cancer in individuals up to ages 50 and 60, respectively (95). Identification of this susceptibility gene would have a significant impact on screening for early lung cancer development, smoking cessation efforts, and chemoprevention trials.

Attention has been paid to polymorphic genes coding for Phase I (mono- oxygenation) and Phase II (conjugation) that function in the activation and detoxification of carcinogens in tobacco smoke. Cigarette smoke contains a mixture of carcinogens, including a small dose of polycyclic aromatic hydrocarbons (PAHs) and 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) among other lung carcinogens, tumor promoters and co-carcinogens (96). Carcinogens such as PAHs and NNK require metabolic activation to exert carcinogenic effects. For instance, Phase I cytochrome P450

15 enzymes oxidize crude PAHs into DNA binding dio-epoxide metabolites. On the other

hand, Phase II enzymes including glutathione S-transferases (GSTs) detoxify PAH

metabolites thus competing with the activation pathways. Smokers have significantly

higher level of DNA adducts from PAHs in the lung compared with nonsmokers (97).

There is some evidence to suggest that people with common polymorphisms of the genes encoding these Phase I and Phase II enzymes may have an increased susceptibility to

lung cancer when exposed to Tobacco (98). However, association studies have yielded conflicting results. It has also been hypothesized that inter-individual variation in DNA

repair and microsatellite instability can modify susceptibility to lung cancer.

1.2.3 Challenges in identification of human lung cancer susceptibility genes

Genetic linkage studies in lung cancer pedigrees and in affected sib-pairs would

allow unambiguous mapping of human lung cancer susceptibility gene(s). However, such

genetic linkage studies have been hindered at least by the following factors:

i) pervasiveness of environmental factors that contribute to lung cancer

development, which will confound the identification of familial clusters of lung cancer

patients;

ii) genetic heterogeneity within population, which means there could be one gene

involved in susceptibility for one family and a different gene involved in susceptibility

for another family;

16 iii) polygenic inheritance of lung cancer, which means more than one genes affect human lung cancer susceptibility as demonstrated in mouse linkage analyses(see below); and

iv) failure in collecting blood samples in eligible families due to poor prognosis of lung cancer patients.

Such limitations have led researchers to perform experimental crossing using inbred strains of mice to genetically dissect lung cancer susceptibility.

1.3 Genetic dissection of lung tumor susceptibility using inbred strains of mice

Inbred strains of mice are produced by 20 or more consecutive generations of brother –sister mating to eliminate genetic variability. Mice within one inbred strain are genetic identical and exhibit same physical and physiological traits, while different strains of mice have different genome make up and often exhibit distinct traits. Using inbred mice to perform experimental crossing can easily produce hundreds of meioses from a single pair of parents, thus eliminating the genetic heterogeneity often associated with human population (99). Because environmental factors are controlled under the same condition, far more complex traits can be probed than is possible for human

17 families (99).

1.3.1 Similarity between human adenocarcinomas (AC) and mouse lung

tumors

Human adenocarcinoma (AC) is the most commonly diagnosed form of lung

cancer in the United States. Of all classes of human lung cancer, the occurrence of AC

seems most subject to a genetic predisposition and least associated with cigarette

smoking (100). Histologically, human AC can be further reclassified into three subtypes:

acinar tumor, mucus-secreting tumors and P-L tumor, a new category combining the

bronchiolo-alveolar carcinoma (BAC) and papillary subtypes (101). The majority of mouse lung primary tumors are histologically early lesions such as hyperplasias and

adenomas. As in the case of human AC, they can progress to invasiveness, nuclear atypia

and metastasis. In structure, histogenesis and molecular characteristics, mouse lung

primary tumors are quite similar to human P-L tumors (102-106).

1.3.1.1 Similarity in anatomical origin of tumors

P-L tumors are positively immunostained for surfactant apoproteins, which are synthesized by both type II and Clara cells, and for a 10-kd Clara cell specific protein,

suggesting that they arise from both type II and Clara cells (102). Lung tumors in mice

grow in a solid form along the alveolar septa, grow as a papillary finger-like projections

18 around a blood vessel or demonstrate a mixed formation. Solid tumors are believed to

arise from type II cells and are of the same lepidic morphology as P-L tumors, while the

cellular origin of papillary tumors is still controversial although their morphology is

similar to Clara cells (102).

1.3.1.2 Similarity in molecular characteristics

Mouse primary lung tumors share molecular characteristics with human P-L

tumors. Kras2 gene mutation is typically associated with human AC as well as mouse

lung tumors. 80 − 90% of mouse lung tumors contained Kras2 activating mutations,

including early hyperplastic lesions (107). Same as those in human AC, mouse Kras2

gene mutations also occur at codons12, 13 and 61. Overexpression of Myc gene without

amplification was also observed (108). LOH studies demonstrated the consistent loss of

regions of mouse chromosome 4 in some mouse lung adenocarcinoma cell lines, and

further investigations demonstrated that this region was syntenic with human 9p21-22, which contains human p16Ink4a gene (109, 110). p16Ink4a deletion or reduced expression

through promoter methylation has been demonstrated in 50% of the adenocarcinomas,

but not in earlier lesions (111). p53 mutation is not observed in hyperplasia and only

rarely in adenoma, suggesting that it is a more malignant characteristic (112). Other

tumor suppressor genes whose expression decreases in both mouse lung tumors and human AC include Rb, Apc, Mcc and Fhit.

19 1.3.2 Inbred strains of mice show different lung tumor susceptibility.

The most useful trait leading inbred strains of mice to be utilized in genetic

dissection of lung cancer is that they show remarkable variety in susceptibility to

spontaneous and chemical-induced lung tumors. Spontaneous lung tumors develop in

approximately 3% of wide mice (113), but inbred strains vary from high risk groups with

100% spontaneous incidence to resistant groups with rare lung tumor development (114)

(Table 1.1). The A/J mouse is one of the most susceptible strains, whereas the C3H,

C57BL/6 mice are among the most resistant to lung tumor development. Other strains, like BALB/c, demonstrate intermediate lung tumor susceptibilities.

Besides incidence, tumor multiplicity and size are other indicators of lung tumor susceptibility. Highly susceptible strains develop more lung tumors than relatively resistant strains. One important aspect is that mouse strains more susceptible to spontaneous lung tumors are also more sensitive to chemical-induced lung tumor development (115), which allows the chemical carcinogens to be used in quantitative tumorigenesis assays such as linkage analysis.

1.3.3 Mouse lung tumor susceptibility governed by Pulmonary adenoma

susceptibility /resistance genes

The variety in lung tumor susceptibility across inbred strains of mice has led

researchers to investigate the underlying genetic factors. Genetic analyses have

20 demonstrated that both mouse lung tumor multiplicity and lung tumor size are continuous

quantitative traits and are affected by quantitative trait loci (QTLs) located in multiple

mouse chromosomes (Table 1.2, Table 1.3). Analyses of lung tumorigenesis in

recombinant inbred strains (RI) between A/J and C57BL/6 mice suggested that there

were three QTLs contributing to the observed differences in susceptibility to lung tumor

development. Malkinson et al. have called these loci as “Pulmonary adenoma

susceptibility” (Pas) genes (116). Statistical analyses of tumor data from such recombinant mice indicated that one of the Pas loci was a major contributor to susceptibility whereas the other two Pas genes were minor contributors. Ryan later found

that a genetic polymorphism at the Kras2 locus on chromosome 6 was strongly associated with susceptibility in these RI strains (117). This was supported by Gariboldi’s

(A/J x C3H) F2 genetic analysis, which mapped the major Pas locus to a 35cM distal region of mouse chromosome 6, named as the Pas1 locus (118). Subsequent linkage studies have confirmed that the Pas1 locus plays a major role in mouse lung tumor susceptibility. For instance, Festing et al. showed the Pas1 locus accounted for a 59.76%

of the total variation in lung tumor susceptibility (119). Devereux used a (A/J x

C57BL/6) F1 x C57BL/6 mouse backcross population and found the Pas1 locus

accounted for about 15% of the total variance (120).

Other Pas loci shown to positively modulate the effect of Pas1 have also been

mapped to mouse chromosomes. Pas2 locus was mapped to chromosome 17 in (A/J

21 xC57BL/6) F2 population (119). It accounted for 7% of the total variance in phenotype.

The human syntenic region for Pas2 is in 6p21 and possible candidates at this location

are the genes for tumor necrosis factor TNF α and β. Another locus Pas3 was mapped to

chromosome 19 and accounted for 3% of the phenotypic variation in the study on (A/J x

C57BL/6) xC57BL/6 backcross mice and 2% of the phenotypic variation when (A/J x

C57BL/6) F2 mice were used (119, 120). Using (A/J x C57BL/6) F2 mice, another Pas

locus, Pas4 was detected in Chromosome 9 accounting for 4% of total phenotypic variation (119).

In addition to Pas loci, the tendency of inbred strains of mice to develop lung

tumor is also affected by several Pulmonary adenoma resistance (Par) loci (Table 1.3).

The Par1 locus was mapped in (A/J x M.Spretus) x C57BL/6 mice to chromosome 11 with a maximum LOD score of 5.3 and accounted for 23% of phenotypic variance under the Pas1/+ background (121). A major resistance locus, designated as Pulmonary adenoma resistance 2 (Par2), has been mapped to the mouse chromosome 18 independently by several groups and accounts for 15% ~40% phenotype variance (122-

124). It has been suggested that the relative resistance of BALB/c mice is due to the interaction between the Pas1 QTL and Par2 QTL in BALB/c mouse genome (125).

Another two Par loci, Par3 and Par4 have also been mapped to mouse chromosome 4 and in (A/J x BALB/c) F2 and (SM x A) RI mice, respectively (124,

126).

22 Counteracting interactions among different susceptibility loci may mask their

individual effects. Using recombinant congenic (RC) strains, Fijneman and Tripodis et al mapped a set of lung tumor susceptibility genes, which they named as “Sluc” for

“Susceptibility for lung cancer”(127-130). Some of Sluc loci overlap with Pas loci but

most of them are novel. Their results further demonstrate that lung cancer is a multigenic

disease, interactions of tumor susceptibility genes are frequent and that they probably

form complex networks.

1.4 Positional cloning of lung tumor susceptibility/resistance genes

Human and mouse genome projects have revealed that mouse and human

genomes share extraordinary homology (131). Positional cloning of mouse lung tumor

susceptibility/resistance genes may ultimately lead to the discovery of their human

counterparts.

Traditionally, gene cloning can be achieved by the following three strategies:

functional cloning, candidate gene approach and “pure” positional cloning (132).

Functional cloning relies on the fundamental information about the basic biochemical defects to identify disease genes. This method does not need chromosome map position information. A well-known example is sickle cell anemia gene cloning. Like the

23 functional cloning, candidate gene approach depends on partial functional information

about the disease genes, although the information may not need to be precise and

complete. A good example is the identification of p53 gene in Li-Fraumeni syndrome

(133). No map information is needed. Different than the above two approaches, the

“pure” positional cloning locates responsible genes solely based on their map positions

while gene functions usually are unknown. With this approach, linkage analyses of

multiple affected families are usually the first step. Cytogenetic DNA rearrangements or

trinucleotide repeat expansion can greatly assist the low- and high-resolution mapping,

such as in gene identification for retinoblastoma and fragile X syndrome (134, 135).

Without such visible cytogenetic changes, positional cloning is still a laborious work.

On the other hand, stimulated by sequence information derived from multiple

genome projects coupled with enriched mouse and human transcript maps, positional cloning strategy has now evolved from its initial “pure” form into the so-called

“positional candidate approach” (132, 136). The positional candidate approach relies on a

combination of initial correct gene mapping and following candidate gene screening. One example is the identification of DNA mis-match repair genes in hereditary non-polyposis

colon cancer (HNPCC). In this case, the HNPCC locus was first mapped to chromosome

2p by linkage analyses (137). Phenotypic information on DNA instability from affected

patients finally led to identification of multiple DNA mismatch repair genes involved in

HNPCC (138-141).

24 Without a clear clue of what candidate genes should be, however, positional

candidate approach will be still laborious and time consuming due to the large number of

candidate genes. Thus, a practical positional candidate approach should include a fine-

mapping step. In human, linkage disequilibrium (LD) and chromosomal haplotype

analyses are often employed to fine map disease loci(142-144). In mice, since we can

experimentally manipulate them, more structured approaches are available now.

1.4.1 Fine-mapping strategies for mouse gene cloning

Genetic analyses on F2 and backcross mouse populations have been commonly

used to map quantitative trait loci (QTL) into chromosome regions. Limited by the

number of recombination events occurring in meioses, however, analyses on these

populations usually turn out relatively broad confidence intervals (CI) and cannot provide sufficiently high resolution for further positional cloning. In order to overcome this obstacle, different experimental designs have been brought up (145).

One way to improve resolution is to perform selective phenotyping on an increased size of mouse population (145). With this method, although phenotyping number is saved, producing such a large mouse population is still impractical for most research laboratories. Other strategies are designed to increase recombination events at the region of interest, which include Advanced Intercross Lines (AILs), recombinant

progeny testing (RPT), interval-specific congenic strains (ISCS), and recombinant inbred

25 segregation test (RIST). Recently linkage disequilibrium (LD) and heterogeneous stock

(HS) were also used to fine map quantitative trait loci (146-149).

1.4.2 Fine-mapping strategies used in present studies

In our laboratory, we have selected congenic strategy to fine map mouse

Pulmonary adenoma susceptibility 1(Pas1) and Pulmonary adenoma resistance 2 (Par2)

QTL. In addition, a newly developed AIL mouse population has also been utilized to

narrow down Pas1, 2, 3 candidate region.

1.4.2.1 Congenic strategy

Congenic strains of mice are produced by continuously backcrossing a donor

strain that harbors a gene or genomic region of interest with a recipient inbred strain.

According to Mendelian law, in a backcross, averagely half of the unrelated genomic

material will be transmitted to a subsequent backcross generation. Theoretically, after ten

generations of backcrosses, the offspring mice will possess approximately 99.90% recipient strain’s genome and carry an interest gene or region from the donor strain (150).

After a congenic strain has been established, further fine mapping may be achieved by generation of subcongenic strains that carry various QTL candidate regions. By careful examination of phenotypes and identification of overlapping QTL region from different subcongenic strains, the original QTL region can be narrowed down. It is noteworthy that

26 while this traditional method of congenic production will take 2.5-3.0 years to complete, a marker-assisted “speed congenic” strategy has been brought up to save time and resources (150, 151).

1.4.2.2 Advanced Intercross Line (AIL) mouse population

An AIL is produced by semi-randomly and sequentially intercrossing a F1 population that initially originated from a cross between two inbred strains (152). A major distinction between producing an AIL and a F2 population is the generation number of intercrossing. With continual intercrossing, the recombination events within a defined chromosome region will be increased. Consequently, the genetic length of the entire genome is stretched and mapping resolution is increased. On the other hand, AIL strategy can also be used for fine mapping of multiple QTLs at the same time and some linked QTLs can be readily separated using an AIL system. In collaboration with

International Livestock Research Institute (Nairobi, Kenya), we have utilized a F11 generation of AIL mouse population to successfully fine map Pas1 QTL (Chapter 1).

1.4.3 Mouse genome project and gene cloning

In April 2001, Celera Genomics produced its mouse genome map after taking the whole-genome-shotgun (WGS) assembly strategy. On the other hand, the public Mouse

Genome Sequencing Consortium (MGSC) used a different assembly strategy, called

27 hierarchical shotgun (HS) assembly, to produce its own mouse genome map. Along with

other genomic information resources such as the Expressed Sequence Tags (ESTs) and full-length mouse cDNA databases (153, 154), these mouse maps are expected to play a more and more important role in gene positional cloning.

At least in three immediate ways enriched mouse genome information can accelerate gene cloning with positional candidate approach. Firstly, on-hand mouse

genome maps set researchers free from developing their own YAC and BAC contigs for the regions of interest, which has been demonstrated as a rate-limiting step in positional

cloning. Secondly, coupled with generation of genome maps, annotation of genomes has

helped provide transcript maps for certain candidate regions. Various techniques were

previously employed to achieve this aim, such as exon-trapping, direct cDNA

hybridization, or cDNA selection. All of them are time-consuming. With human and

mouse genome sequencing projects accomplished, computational analysis on the genome

sequence data, which generally characterized with programme-dependent gene prediction

and similarity-based database search using BLASTN/X or other tools, has become a

direct and effective way to identify a potential gene. A third impact is that through

computational analyses of single nucleotide polymorphisms (SNPs), the genes bearing

functional SNPs can be prioritized for experimental analyses. Thus, time and resources

can be enormously saved.

Although bioinformatics is extremely important, however, it is also noteworthy

28 that the experiment activity is still an indispensable part and is the final resort in gene identification. Most of time, for instance, computer-derived gene predictions only produce partial transcript sequences. Either library screening, 3’-/5’- RACE assays or other experimental techniques will be still needed to generate full-length transcript sequences. On the other hand, SNPs produced by computer programs should also be confirmed by experiments.

1.5 Objective and summary of the study

Inbred mouse strains show widely different susceptibilities to both spontaneous and chemical-induced lung tumorigenesis and present a useful model system for lung cancer genetics research. Genetic analyses have revealed that mouse lung tumor susceptibility is a quantitative trait and genetically governed by both Pulmonary adenoma susceptibility (Pas) and Pulmonary adenoma resistance (Par) genes. A major susceptibility locus, Pas1, and a major resistance locus, Par2, have been mapped to mouse distal chromosome 6 and chromosome 18, respectively. These two QTLs have been playing a significant role in predisposition to mouse lung tumor development.

Cloning of mouse genes responsible for these QTLs will greatly accelerate the identification of their human counterparts.

29 Our objective for the present studies is that by fine mapping and candidate gene screening, we can identify candidate genes for the Pas1 and Par2 loci. Further

characterization of these candidate genes will lead to the identification of Pas1 and Pas2

genes. Chapter 1 introduces the background knowledge of human and mouse lung

cancer genetics. In addition, positional cloning strategies and impacts of bioinformatics on post-genome gene cloning were also discussed. In Chapter 2, we utilized a F11 generation of advance intercross line (AIL) mouse population to fine map Pas1, 2, 3

QTLs and successfully fine mapped the Pas1 QTL into a ~1.3-Mb (~ 1-cM) candidate

region. In Chapter 3, independently from the AIL project, we generated congenic mice

to fine map the Pas1 locus. The result presented a smaller (~1-Mb) candidate region for

the Pas1 QTL. Together, the results from the AIL project and congenic project provided

us a ~1-Mb minimum Pas1 candidate region. Candidate gene screening thus became

practical. In Chapter 4, candidate gene screening was performed in the minimum Pas1

candidate region. Nucleotide polymorphism and gene expression analyses have presented three candidate genes for the Pas1 QTL (i.e. Lrmp, Pas1c1 and Pas1c2). In Chapter 5, we have used similar congenic strategy to fine map the Par2 QTL. Our results generated a 6.3-cM Par2 candidate region. Expression analyses using real time RT-PCR technology revealed several Par2 candidate genes exhibiting expression differences between A/J and

BALB/c mice. The Rad30b gene bears 25 nucleotide polymorphisms in its coding region between A/J and BALB/c mice, causing ten amino-acid alterations. Finally, in Chapter

30 6, future directions for Pas1 and Par2 gene cloning were briefly addressed.

31

% of animals with Mean tumor multiplicity Mouse inbred spontaneously occurring /mouse after carcinogen Strains tumors at 18 months of age induction A/J and A/HeJ 90 20-25 SWR/J 90 15-20 BALB/cJ 40-50 3-4 CBA/J 20 1.1 129/RrJ NR 2-3 DBA/2J 5-10 0.2-0.4 C3H/HeJ 5-10 0.1 SJL/J 5 0.3 AKR/J 5 0.1 C57L/J 1 0.1 C57BL/6J 1 0.1

Table 1.1 Variant pulmonary adenoma susceptibility in inbred strains of mice. Adapted from Table IV, Malkinson A.M., Toxicology, 1989 (reference 114).

32

Position Named Chr. Genetic marker Experimental cross Author (reference) (cM) loci 1 82 D1Mit33 A x BALB/c F2 Festing et al. (124) 4 42 D4Mit77 A x BALB/c F2 Festing et al. (124) 6 37 D6Mit263 A x BALB/c F2 Festing et al. (124) A x C3H F2 Garibodi et al. (118) (A x C57BL/6) x C57BL/6 Devereux et al. (120) 6 71 Kras2 Pas1 A x C57BL/6 F2 Festing et al. (119) (A x M.Spret.) x C57BL/6 Manenti et al (157) 9 47 D9Mit11 A x C57BL/6 F2 Pas4 Festing et al. (119) 10 21 D10Mit126 A x B, B x A RI Lin et al. (158) 13 36 D13Mit279 A x BALB/c F2 Festing et al (124) Tnfb (Lta) Ax C57BL/6 F2 Festing et al. (119) 17 19 Pas2 H2 A x B, B x A RI Lin et al. (158) 5 D19Mit42 (A x C57BL/6) x C57BL/6 Devereux et al. (120) 19 Pas3 15 D19Mit16 A x C57BL/6 F2 Festing et al. (119) 19 46 D19Mit10 A x B, B x A RI Lin et al. (158)

Table 1.2 Pulmonary adenoma susceptibility (Pas) loci in mouse genome.

Position Named Chr. Genetic marker Experimental cross Author (reference) (cM) loci 6 3 D6Mit50 SWR x BALB/c F2 Par4 Manenti et al. (123) 56 D11Mit54 A x Mus Spretus F2 Manenti et al. (121) 11 A x BALB/c F2 Par1 Festing et al. (124) 54 D11Mit70 A x SM RI Pataer et al. (126) 12 38 D12Mit5 A x SM RI Par3 Pataer et al. (126) 41 D18Mit9 SWR x BALB/c Manenti et al. (123) 18 44 D18Mit103 (A x BALB/c) x BALB/c Par2 Obata et al. (122) 47 D18Mit188 A x BALB/c F2 Festing et al. (124)

Table 1.3 Pulmonary adenoma resistance (Par) loci in mouse genome.

33 CHAPTER 2

FINE MAPPING OF PULMONARY

ADENOMA SUSCEPTIBILITY 1,2,3 (PAS1, 2, 3) QTLS

USING ADVANCED INTERCROSS LINE (AIL) MOUSE

POPULATION

2.1 Introduction

Lung cancer is the leading cause of cancer-related death in both the United States

and major industrialized nations (1). Epidemiological studies have indicated that ~85% of

all lung cancer deaths in the United States are associated with tobacco smoking (155).

Tobacco smoking increases the relative risk for lung cancer in smokers by 13-fold and in passive smokers by 1.5 fold (3). Although the majority of lung cancer cases are

associated with cigarette smoking, increasing evidence suggests that individuals differ in

their susceptibility to lung cancer. An increased familial risk for lung cancer was observed among relatives of lung cancer probands (88-94). Further segregation analyses

34 provided evidence that susceptibility to human lung cancer follows a pattern of autosomal

dominant Mendelian inheritance (95). However, there have been no reports on the

localization and identification of human lung cancer susceptibility genes.

Different inbred mouse strains show widely different susceptibilities to both

spontaneous and chemically induced lung tumor formation and thus serve as models for

research in lung cancer genetics (156). The multiplicity of mouse lung tumors is a

quantitative trait controlled by multiple genetic loci (114). Recent linkage studies have

been conducted to identify Pulmonary adenoma susceptibility (Pas) and Pulmonary

adenoma resistance (Par) loci. A major susceptibility locus was mapped using (A/J x

C3H/HeJ) F2 mice to distal chromosome 6, and was termed the Pas1 locus (118). This locus produced a maximum logarithm of the likelihood ratio (LOD) score of 9(118).

Consistent results were obtained in comprehensive linkage studies using (A/J x

C57BL/6J) F2, (A/J x C57BL/6J) x C57BL/6J, (A/J x Mus Spretus) x C57BL/6J, and A x

B & B x A recombinant inbred mice (119, 120, 157, 158). Additional loci shown to

modulate the effect of Pas1 were mapped to chromosomes 17 and 19. Linkage to a locus

on chromosome 17, the site of the putative Pas2 locus, was observed in (A/J x

C57BL/6J) F2 mice (119). The location of the Pas2 locus is homologous to human chromosome 6p21; potential candidates at this location are the genes for tumor necrosis factors α and β. Similarly, linkages to lung tumor susceptibility were also seen at markers on chromosome 19 (Pas3) using (A/J x C57BL/6J) x C57BL/6J mice and (A/J x

35 C57BL/6J) F2 mice (119, 120). Other potential lung tumor susceptibility loci and their

complex interactions have been identified using recombinant congenic strains (127-130).

Most quantitative trait loci (QTL) detection and mapping studies in mice have

been carried out using F2 intercross and backcross designs. Due to limited numbers of

recombination events in small chromosomal regions, the mapping resolution has been

restricted to relatively large confidence intervals (CI). However, precise localization of

quantitative trait loci (QTL) is required for positional cloning. To address this, a number

of population designs have been proposed that increase recombination frequency in QTL-

segregating populations (145). The Advanced Intercross Line (AIL) design is one approach (152). An advantage of this design over congenic approaches is its suitability for simultaneous refinement of multiple loci (159). In addition, the AIL design lends itself to situations where information on the number of QTLs in a particular region is unavailable, since data obtained with advanced intercross lines potentially allows linked

QTLs to be dissected into constituent loci (159).

In this report, we applied the AIL design of higher resolution mapping to further characterize Pas1-3 using the F11 generation of the (A x C57BL/6) AIL mouse

population. Our objective was to fine map the Pas QTLs to a sufficiently small size (<1

cM) for future candidate identification.

36 2.2 Materials and methods

Advanced Intercross Line (AIL)

The F11 generation of the (A x C57BL/6) AIL mice developed at the International

Livestock Research Institute (Nairobi, Kenya) was used in the present study. Original

parental strains, C57BL/6JOIaHsd and A/JOlaHsd (Harlan UK Ltd., Shaws Farm.

Blackthom, Bicester, Oxon, OX6 OTP, U.K.), produced more than 35 F1 litters. We

generated F2 mice by intercrossing 50 pairs of F1 mice; there were no duplicate mating.

Sibling pairing was avoided, but otherwise pairing was random. For each generation after

F2 to F10, 50 litters were produced and 65 breeding pairs selected to produce the following generation. The same strategy that was used in producing the F2 was adopted in

all the subsequent generations. More than 200 pairs of F10 were selected to produce

approximately 1,120 F11 mice.

Lung tumorigenesis study

The F11 AIL mice were used in a mouse lung tumor bioassay. Four- to six-week-

old mice received a single i.p. injection of urethane (1 gm/kg), and 6 months later, mice

were sacrificed by CO2 asphyxiation, and their lungs removed, and fixed in 10% buffered

formalin. Surface lung tumors were counted with the use of a dissecting microscope.

37 Genotype data collection

In total, 1,120 (A x C57BL/6) F11 AIL mice were used in the lung tumor

bioassay. Following counting of lung tumors, genomic DNA of F11 and parental strain

mice was prepared from tails. A tail clipping from each F11 mouse was homogenized and

incubated overnight at 37˚C in lysis solution (pronase 0.4 mg/ml, 10% sodium

dodecylsulfate (w/v), 10 mM Tris, 400 mM NaCl, and 2 mM EDTA) followed by

saturated NaCl extraction, precipitation with ice-cold 100% alcohol, and washing twice

with 70% alcohol. Approximately 200 mice with the highest tumor multiplicity and 200

mice with the lowest tumor numbers were selected for genotyping using all the available

microsatellite markers for regions covering Pas1-3. “Mit” polymorphic markers were

purchased from Research Genetics (http://www.resgen.com/). D6Osu6 marker was

developed from Celera mouse genomic sequence (see Chapter 3). Forward primers were

radioactively end-labeled with γ- P32-ATP (3,000Ci /mmol). The PCR reaction profile

consisted of one cycle at 95˚C for 2 mins, followed by 30 cycles at 94˚C, 55 ˚C, and 72˚C each for 30 sec. The PCR fragments were resolved in 6% denaturing polyacrylamide gels containing urea. After electrophoresis, the gels were dried and exposed to X-ray films overnight.

38

Linkage analyses

A square root transformation was used to normalize the phenotypic data prior to statistical analyses. QTL analyses were performed using three different statistical procedures: 1) MapMaker EXP/QTL software analysis; 2) ANOVA analysis based on a multiple linear regression model including second-order interactions with Pas1 QTL; and

3) single-term multiple linear regression analysis.

MapMakerEXP/QTL analysis Genotypic and phenotypic data were entered into

MapMaker EXP/QTL using map and marker positions published at Jackson Laboratories

(Bar Harbor, ME). Resulting LOD score information was then scaled by linear interpolation from genetic position to physical position according to the Celera CDS 3.6 database (Celera Genomics, Rockville, MD). To examine an apparent interaction between Pas1 and Pas2, data from animals heterozygous at D6Osu6 were stratified and examined for linkage in Pas2.

ANOVA and regression analysis Genotypes were encoded for ANOVA as factor levels with (AA = -1, AB = 0 and BB = 1). A generalized linear multiple regression model including second-order interactions with Pas1 (y ~ . + D6Osu6 * .) was used in an

ANOVA setting to evaluate marker association and interactions. Linear multiple regression using only simple terms (y ~ .) was also performed. In this scenario, marker interactions were omitted.

39 Bioinformatics

The physical position of each marker is derived from Celera Genetics mouse

genome map (CDS3.6) by performing BLASN search with marker sequence. Marker

sequences were obtained from Whitehead Institute /MIT Center for Genome Research

(WICGR)(http://www-genome.wi.mit.edu/). The genetic position of each marker is

derived from Mouse Genome Database (MGD)(http://www.informatics.jax.org/).

2.3 Results

Figure 2.1 shows results from the MapMaker EXP/QTL analysis with markers

placed according to their physical position. For the Pas1 QTL, a peak was found between

the D6Osu6 and D6Mit294 markers with the highest LOD score at D6Mit57 (Figure

2.1a). Extremely high LOD scores (>150) were observed, which can be attributed to the

AIL breeding method, a large gene effect of Pas1 QTL, and/or the selective genotyping

strategy. Iraqi et al. also found large LOD scores in some regions when using AIL to fine

map trypanosomiasis resistance genes (159). They used a 2-LOD supporting interval with

“95 % confidence” citing this as conservative estimation (159). The use of 1- or 2-LOD

supporting intervals as confidence intervals was found not highly informative however in

the present study because the interval would have been placed very close to a single

40 marker, i.e. D6Mit57.

ANOVA analysis of Pas1 QTL has shown that while the markers proximal to

D6Mit57 are highly linked, D6Mit15 is also associated with mouse lung tumor susceptibility (Table 2.1), revealing some complexity within the Pas1 QTL region.

Finally, single term linear regression analysis revealed that only the D6Mit57 marker was significantly associated with lung tumor susceptibility. Thus the confidence interval for the Pas1 QTL could be conservatively defined with the D6Osu6 and

D6Mit294 markers (Figure 2.2a). In the Celera mouse genome map (CDS3.6), these two markers are adjacent to Eca39 and Krag genes, respectively. The genetic distance between these two markers is <1 cM according to Mouse Genome Database or MGD, and the physical distance is ~1.3 Mb.

For the Pas2 QTL, inclusion of all animals showed only an insignificant peak by

MapMaker Exp/QTL analysis. However, when only mice heterozygous at D6Osu6 were included, the peak near D17Mit16 exceeded LOD = 2, suggesting interaction between the

Pas1 and Pas2 QTLs and that dominance of Pas1 could mask the Pas2 effect (Figure

2.1b). This interacting relationship has been reinforced by both ANOVA (Table1) and regression results (Figure 2.2b). The Pas2 QTL lies in the 7.6-Mb region between

D17Mit23 and D17Mit231 with the most likely candidate region localized at D17Mit16 and not extending to D17Mit23.

41 No marker at Pas3 was linked to lung tumor susceptibility (Figure 2.1c, Table 2.1 and Figure 2.2c). The highly non-Mendelian distribution of Pas3 alleles suggests that the breeding process produced selective pressure against the AA genotype. It is not clear how to interpret this pressure since the entire region surveyed was affected.

2.4 Discussion

An AIL is produced by semi-random intercrossing (avoiding sibling pairing) in each generation from F2 onwards, until the desired advanced intercross (03, 04, 05 etc.) is obtained (152). For QTL fine-mapping purpose, individuals in the latest generation are phenotyped and genotyped. In this manner, many recombination events applicable for high-resolution mapping of QTL accumulate in a relatively small population over multiple generations. With the same population size and QTL effect, providing that marker spacing is not limiting, the 95% CI of a QTL map location can be reduced, in principle, by a factor of t/2, where t is the number of advanced generations (152). This reduction can be obtained, in practice, up to the sixth or eighth generation, if each generation is derived from a minimum of approximately 50 breeding pairs from the previous generation (152). So far, only the International Livestock Research Institute

42 (Nairobi, Kenya) research group has developed AILs; they successfully applied this

genetic mouse model to fine map trypanosomiasis resistance QTLs (159).

The goal in this study was to narrow down the Pas1-3 loci to eventually identify

the Pas gene(s) mapped by QTL approach. We successfully resolved Pas1 QTL into a

~1.3 Mb physical region defined by the markers D6Osu6 and D6Mit294. The highest

LOD value appearing at the D6Mit57 marker confirmed previous genetic analyses, which

has shown that the Pas1 locus is tightly linked to the Kras2 gene. The Pas2 QTL has

been detected by both ANOVA and regression analyses but not by Mapmaker software.

Analyses revealed an interaction between Pas1 and Pas2. However, we did not detect any significant QTLs at locations on Chr19 where Pas3 would be expected, presumably due to the diminution of Pas3 QTL caused by loss of one of the alternative alleles in the process of breeding. The etiology of the selective pressure against Pas3 AA genotype is unclear. While AIL mapping can considerably improve QTL resolution, the present study also indicates that, in spite of care in randomizing mating and maintaining effective population size, some weak QTL alleles may be lost in the population development.

The Pas1 QTL is a major susceptibility locus based on the genetic linkage studies in several crosses as reviewed in the previous section (118-120, 157, 158). This locus accounts for approximately 50% of the observed phenotypic variance, indicating that the

Pas1 plays a major role in inherited predisposition to lung tumor development in mice.

More importantly, recent studies have demonstrated that there is a significant association

43 between a Kras2/RsaI polymorphism (located within the Pas1 locus) and the risk and prognosis of lung adenocarcinoma in both Italian and Japanese populations in case- control studies (160, 161). These results suggest that the Pas1 gene may also play an important role in the susceptibility to human lung cancer. The candidate region produced from this study, together with the one from the congenic strategy (see Chapter 3), has helped us establish a minimum Pas1 candidate region for candidate gene screening.

44

Figure 2.1 MapmakerEXP/QTL analyses on Pas loci. Results were scaled according to the physical position of markers on the Celera genome database CDS 3.6 (also see legend for Fig.2.2). Solid line shows result using data for all animals. Dotted line shows result using data from animals heterozygous at D6Osu6.

45

46 Figure 2.2 Linear multiple regression analysis of Pas loci. Markers are placed at physical positions. Results are plotted as 1 – p value so that the top is most significant.

Dotted line marks p = 0.05. A. Results for Pas1. Markers were aligned from left to right:

D6Mit59, Pik3c2g, Sox5, D6Osu6, D6Mit57, D6Mit294, D6Mit15, D6Mit201,

D6Mit373, D6Mit26, D6Mit372, D6Mit390. B. Results for Pas2. Markers were aligned from left to right: D17Mit 23, D17Mit16, D17Mit231, D17Mit13, D17Mit11, D17Mit10.

C. Results for Pas3. Markers were aligned from left to right: D19Mit42, D19Mit86,

D19Mit132, D19Mit19, D19Mit89, D19Mit53, D19Mit10, D19Mit37.

47

48 P value QTLs Markers AA n AB n BB n Main x D6Osu6 Effect D6Mit59 5.4 107 3.8 163 1.7 129 < 2.2e-16* Pik3c2g 5.5 108 3.9 153 1.7 138 < 2.2e-16* Sox5 5.8 102 4.4 130 1.6 167 < 2.2e-16* D6Osu6 5.8 109 4.4 133 1.3 157 < 2.2e-16* D6Mit57 5.7 120 4.3 124 1.3 155 < 2.2e-16* D6Mit294 5.7 116 4.3 133 1.3 150 0.106 Pas1 D6Mit15 5.7 117 4.3 132 1.3 150 0.005* D6Mit201 5.7 118 4.3 131 1.3 150 0.159 D6Mit373 5.7 119 4.2 131 1.3 149 0.948 D6Mit26 5.7 106 4.4 141 1.3 152 0.745 D6Mit372 5.7 105 4.4 142 1.3 152 0.075 D6Mit390 5.7 105 4.3 145 1.3 149 0.306

D17Mit23 3.6 110 3.6 192 3.4 97 0.306 0.085 D17Mit16 3.9 128 3.4 193 3.3 78 1.6e-06* 0.003* D17Mit231 4 123 3.4 173 3.4 103 0.489 0.007* Pas2 D17Mit13 4 109 3.4 205 3.4 85 0.495 0.679 D17Mit11 3.7 138 3.5 188 3.5 73 0.018 0.747 D17Mit10 3.5 160 3.6 159 3.7 80 0.254 0.092

D19Mit42 3.2 39 3.4 199 3.8 161 0.687 D19Mit86 3.2 46 3.5 212 3.8 141 0.274 D19Mit132 3.4 22 3.4 182 3.8 195 0.975 D19Mit19 3.6 44 3.7 176 3.5 179 0.795 Pas3 D19Mit89 3.3 50 3.6 182 3.6 167 0.797 D19Mit53 3.3 54 3.7 149 3.5 196 0.500 D19Mit10 3.3 56 3.8 174 3.4 169 0.192 D19Mit37 3.3 63 3.6 210 3.6 126 0.093

Table 2.1 ANOVA analysis on Pas QTLs * indicates it is statistically significant (P<0.01)

49 CHAPTER 3

FINE MAPPING OF PULMONARY

ADENOMA SUSCEPTIBILITY 1(PAS1) QTL USING

CONGENIC MICE

3.1 Introduction

Lung cancer is the leading cause of cancer deaths in the United States (1). The vast majority (80-90%) of lung cancer cases are associated with tobacco smoking (2).

Relative risk for lung cancer is increased in smokers at least 13 fold and in passive smokers by 1.5-fold, with a linear relationship between the number of cigarettes smoked and lung cancer risk (3, 162). Other environmental and occupational exposures to asbestos, arsenic, chromium, radon etc also increase risk to develop lung cancer (5-7).

Although environmental factors represent the main risk factors for lung cancer, genetic components may also play an important role. Familiar clusters have been observed on lung cancer patients (88-94). Further segregation analyses provided evidence

50 that susceptibility of the human population to different forms of lung cancer follows a

pattern of autosomal dominant Mendelian inheritance (95). Genes involved in the courses

of activation and detoxification of tobacco carcinogens have been intensively investigated. However, the results often turn out contradictory (14). On the other hand,

the pervasiveness of lung carcinogens in our environment and poor prognosis associated

with the disease has made it difficult to accurately identify susceptibility genes in human

familiar clusters.

Lung tumors in mice represent an excellent model system for the study of the

interactions between genetic and environmental factors that lead to tumor formation.

Inbred strains of mice have different susceptibilities to spontaneous and carcinogen-

induced lung tumor formation (114). The A/J strain is the most susceptible to lung

tumorigenesis whereas the C3H and C57BL/6 strains are the most resistant. In the past

two decades, genetic studies on inbred strains of mice have revealed that both mouse lung

tumor size and multiplicity are quantitative traits and are genetically governed by

multiple quantitative trait loci (QTLs) in the mouse genome. One of these QTLs,

Pulmonary adenoma susceptibility 1 (Pas1) has been mapped to mouse distal

chromosome 6 and plays a major role in predisposition to mouse lung tumor development

(118-120, 157, 158).

One major obstacle towards positional cloning of the Pas1 QTL is to refine the

candidate region. Previous genetic studies using either F2 or backcross mice have

51 localized the Pas1 QTL on the mouse distal chromosome 6 but unanimously generated a

relatively large region that is not suitable for positional candidate approach. Recently,

congenic strategies have become increasingly important for QTL analyses (163). In the present study, we targeted an approximately 26.1cM(~36Mb) of region on the mouse distal chromosome 6, which had been demonstrated to harbor the Pas1 QTL before, to generate congenic mice using A/J and C57BL/6J inbred strains. Fine mapping was performed on multiple subcongenic mice containing various fragments of A/J allele. The overlapping QTL-containing regions resulted in a less than 1Mb of QTL region.

3.2 Materials and methods

Construction of congenic and subcongenic mice

The flowchart of experimental design has been demonstrated in Figure 3.1. Inbred

A/J and C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor,

ME). Animals were housed in plastic cages with hardwood bedding and dust covers, in a

HEPA filtered, environmentally controlled room (24 ± 1oC, 12/12 hr light/dark cycle).

Animals were given Rodent Lab Chow, #5001 (Purina) and water ad libitum. The basic

breeding scheme in the study was to put an approximately 26.1 cM fragment of

chromosome 6, encompassed by D6Mit54 and D6Mit373 markers from lung tumor

52 susceptible A/J strain onto the genetic background of lung tumor resistant C57BL/6J

mice. Therefore, selection of this fragment governs the choice of mice for mating. A/J

mice were initially crossed to C57BL/6J mice. F1 progeny were backcrossed to

C57BL/6J mice to produce the first backcross generation (N2). N2 heterozygous for the

chromosome region of interest were then backcrossed again to C57BL/6J mice to

produce the N3 generation. This process was repeated for a total of nine backcrosses. At

N8, 132 male subcongenic strains containing different chromosomal fragments were

generated. The individual subcongenic strains were then each crossed to three C57BL/6J

females to produce the N9 generation for lung tumor bioassay.

Genotyping using polymorphic markers

In order to select mice on the basis of their genotypes throughout the region of

interest on chromosome 6, the following markers were used: D6Mit54, D6Mit52,

D6Mit59, D6Mit57, D6Mco10, D6Mco11, D6Mit15, and D6Mit373. For DNA isolation

tail clippings from each mouse were homogenized and incubated overnight at 37oC in

nucleic acid lysis solution (Pronase 0.4 mg/ml, 10% sodium dodecylsulfate (w/v), 10 mM

Tris, 400 mM NaCl, and 2 mM EDTA) followed by saturated NaCl extraction,

precipitation with ice-cold 100% alcohol and washing twice with 70% alcohol. The

mouse microsatellite primers were purchased from Research Genetics, Inc. (Huntsville,

AL). The forward primer was end-labeled with γ-32P-ATP, and 30 cycles of PCR were

53 performed at 94°C for denaturation, 55°C for annealing and 72°C for extension. Eight percent denaturing polyacrylamide gels were used for resolution of the radiolabeled PCR products followed by autoradiography.

Polymorphic markers between D6Mit59 and D6Mit15

Based on Celera mouse genome sequence (CDS 3.6), polymorphic microsatellite markers were developed in the Ohio State University and designated with “D6Osu”.

Total 53 microsatellite DNA fragments in the region encompassed by D6Mit59 and

D6Mit15 markers were initially selected. Oligonucleotide primers were designed for each fragment and length polymorphism was examined using A/J and C57BL/6J genomic

DNA. Single nucleotide polymorphisms (SNPs) were derived from direct sequencing results. “Bcat1” marker is derived from 2-bp deletion in Bcat1 gene promoter region.

“Mco” markers were developed in the Medical College of Ohio and are located within

Kras2 gene intron or its near region.

PCR- Single-stranded conformation polymorphism (PCR-SSCP) analysis

For single nucleotide polymorphisms (SNPs), we performed PCR-Single-Strand

Conformation Polymorphism (PCR-SSCP) analyses. Briefly, 1 µl of purified PCR product (about 5 ng of DNA) was end-labeled with γ-32P- ATP and was loaded onto a 6% non-denaturing polyacrylamide gel. Electrophoresis was performed at 30 W for 20 hours

54 at 4oC. The gel was then transferred onto filter paper, dried and exposed to X-ray film for

1-2 hours at room temperature.

Lung tumor bioassay on congenic mice

Five-week-old N9 mice were given a single i.p. injection of urethane (1 mg/g body weight) in 0.2 ml PBS. All animals were euthanized by CO2 asphyxiation 7.5

months after urethane initiation. A portion of lung tumors and normal tissue were

removed and flash frozen in liquid nitrogen. The rest of lungs were fixed in

Tellyesniczky’s solution and examined with the aid of a dissecting microscope to obtain

the tumor count and size. Tumor volumes were determined by measuring the three-

dimensional size of each tumor and by using the average of the three measurements as

the diameter. The radius (diameter/2) was determined, and the total tumor volume was

calculated by: Volume = (4/3)πr3 (r-radius).

Statistical Analysis

One-way ANOVA was used to determine the difference in the number of

pulmonary tumors per mouse between control and treated groups. Two-way ANOVA

was used to determine the difference in both the number and the size of lung tumors

between control and treated groups.

55 3.3 Results

Congenic and subcongenic strains

The exact region of the Pas1 QTL that was selected to construct congenic mice

was illustrated in Figure 3.2. The genetic distance covering the Pas1 QTL is

approximately 26.1 cM encompassed by D6Mit54 and D6Mit373 markers in Mouse

Genome Database (MGD, Jackson Laboratory). In the Celera mouse genome map

(CDS3.6), D6Mit54 and D6Mit373 markers are located at Contigs GA_x54KRFPKN04 and GA_x6K02T2NTSL respectively, which define a physical distance of ~36 Mb. Pas1

congenic mice were constructed by transferring this ~36-Mb chromosome 6 fragment

from the A/J (donor) mice onto the genetic background of the resistant C57BL/6J mice

(recipient) by a total of nine backcrosses. According to Mendelian genetic law, averagely

half of the unrelated genomic material will be transmitted to a subsequent backcross

generation in a backcross. Thus in our system, after nine backcrosses, averagely 99.81%

of genetic components in a subcongenic strain mouse is expected to be of C57BL/6J-

derived and only 0.19% is from the A/J parental strain (150). At N5 generation,

additional microsatellite markers on chromosomes 9, 10, 17, and 19, including D9Mit75,

D9Mit355, D9Mit35, D10Mit106, D10Mit2, D10Mit126, D17Mit246, D17Mit23,

D17Mit50, D19Mit36, D19Mit10, and D19Mit89, were screened to obtain the optimal

breeders that harbor the least amount donor genome. After seven generations of

56 backcrosses, 20 male N7 congenic mice that carried the A/J donor region (defined by

markers D6Mit54 and D6Mit373) were further backcrossed to C57BL/6J females. Mice

with crossovers in various places throughout the region were selected as the N8

subcongenic strains. A total of one hundred thirty two male subcongenic strains (N8) that

contained various quantitative trait locus (QTL) subregions were produced from such

crossover mice. All the mice have been genotyped at eight microsatellite markers,

D6Mit54, D6Mit52, D6Mit59, D6Mit57, D6Mco10, Mco11, D6Mit15, and D6Mit373 to

define the chromosomal segment from the A/J mice into the C57BL/6J background in

each subcongenic strain.

Lung tumorigenesis of congenic mice

Each congenic or subcongenic strain was crossed with 3 female C57BL/6J mice.

The urethane-induced lung tumor bioassays were carried out on the N9 generation of

mice. The entire 26.1 cM of genome fragment substituted in the congenic strain (AB)

with the A/J allele is heterozygous for the markers located within the region. The

urethane-induced lung tumor multiplicity and total tumor volume in congenic mice

showed a significant difference between the congenic strain and corresponding control

strain (Table 3.1). An average of 6.0 tumors/mouse with total tumor volume 1.91 mm3 were observed in the congenic strain (AB), while an average of 0.7 tumors/mouse with total tumor volume 0.18 mm3 were seen in the control mice (BB) (P<0.0001). Thus the

57 substitution of one A/J allele for C57BL/6J in the Pas1 region increased the mouse lung tumor susceptibility by ~8.6-fold in tumor number, ~10-fold in total tumor volume.

Strains 1 through 8 represent subcongenic strains containing various donor fragments as indicated. If the donor fragment carries the Pas1 locus, then the subcongenic strain will show the high lung tumor susceptibility observed in the congenic strain (AB). If the Pas1

A/J allele is absent in the donor fragment, the subcongenic strain carrying it may have the similar tumor susceptibility as the control strain (BB). The Pas1 QTL was then narrowed to a small region of approximately 7.4-Mb between the markers D6Mit59 and D6Mit15 based on the combined analysis of the phenotypic results from the N9 mice and the genotypes of substituted chromosomal fragment (Figure 3.3). We have forty-eight subcongenic strains carrying a recombination between the markers D6Mit59 and

D6Mit15. In order to further refine the region that contains the Pas1 QTL, development of polymorphic markers between D6Mit59 and D6Mit15 become essential.

Development of markers and fine-structure mapping of the Pas1 QTL

Based on the public database and the Celera database, sequence analysis was performed and eighteen new polymorphic markers within the Pas1 QTL region between

D6Mit59 and D6Mit15 were developed between A/J and C57BL/6J strains (Table 3.2).

Two types of markers have been developed. One type is based on single nucleotide polymorphisms (SNPs), which were resolved by PCR-SSCP. The other group is

58 microsatellite markers, whose PCR products were resolved either by agarose gel electrophoreses or polyacrylamide gel electrophoreses (PAGE). Using these new markers, we have further narrowed the Pas1 QTL to a ~1-Mb physical region defined by the markers D6Osu6 and D6Osu11. All of the new markers were located in the Celera mouse genome scaffold GA_x6K02T2NTSL, which was constructed with 22 overlapping

BAC tiles in a total length of 15,827,304 base pairs. The D6Osu6 marker is adjacent to the Bcat1 gene while D6Osu11 marker is near the Krag gene. All the genes beyond this region encompassed by the Bcat1 and Krag were excluded as candidates for the Pas1 gene(s) based on congenic strategy. There are twenty-four putative genes were identified between the markers D6Osu6 and D6Osu11 including several known genes: branched chain aminotransferase 1, cytosolic (Eca39Bcat1), lymphoid-restricted membrane protein

(Lrmp/Jaw1), Kras2, tubulin alpha 7 (Tuba7), basic helix-loop-helix domain containing, class B (Bhlhb2l-pending /Dec2) and nineteen unknown transcripts (Table 3.3).

3.4 Discussion

Previous linkage study using (A/J × C3H/HeJ) F2 mice has demonstrated that the

Pulmonary adenoma susceptibility 1(Pas1) QTL is the major mouse lung tumor susceptibility locus that accounts for approximately 50% of the phenotypic variance and

59 has mapped the locus to the distal region of mouse chromosome 6 (118). Consistent

results were obtained in independent studies using (A/J × C57BL/6J) F2 (60% variance),

(A/J × C57BL/6J) × C57BL/6J (16% variance), (A/J × M. spretus) × C57BL/6J (34%

variance), and A × B & B × A RI mice (51% variance) (118-120, 157, 158). In order for

positional cloning of the Pas1 gene(s), however, efforts are needed to fine map the Pas1

QTL into a small candidate region.

So far two fine mapping strategies have been used to fine map the Pas1 QTL.

Fine mapping by linkage disequilibrium (LD) has been performed by Manenti et al to

narrow down the Pas 1 candidate region to a ~1.5Mb candidate region (146). In Chapter

2, genetic analysis on a F11 generation of AIL mouse population has enabled us to fine

map the Pas1 QTL into a ~1.3 Mb region encompassed by the D6Osu6 and D6Mit294

markers.

In the present study, we have utilized congenic mice to further refine the Pas1

QTL. Consistent with previous studies(107, 118, 125), our results demonstrated that the

Pas1 locus is associated with susceptibility to mouse lung tumor development after

urethane treatment, both in tumor multiplicity and in tumor size. A semi-dominant A/J

Pas1 allele contributed approximately 8.6-fold differences in lung tumor multiplicity,

~10-fold in tumor load between the congenic strain (AB) (6.0 tumor/mouse, 1.91 mm3 tumor load) and control strain (BB) (0.7 tumor/mouse, 0.18 mm3 tumor load) mice.

60 By crossing the congenic strain (AB) with parental strain C57BL/6J, one hundred

thirty two subcongenic strains (N8) containing various donor fragments in the congenic

region were generated. Development of new polymorphic markers in the congenic region

has allowed us to refine the Pas1 QTL into a less than 1 Mb candidate region defined by

the markers D6Osu6 and D6Osu11.Compared to the candidate regions derived from AIL

and linkage disquilibrium (LD), the region defined by the D6Osu6 and D6Osu11 markers

is the minimum candidate region for the Pas1 QTL at this moment.

The Kras2 gene has long been considered as a major Pas1 candidate and is

located in the minimum candidate region. The activation of the Kras2 gene has been

revealed as an early event frequently found in both spontaneously occurring and

chemically induced mouse lung tumors (107). Polymorphisms detected in the Kras2

transcription regulatory regions in different mouse strains correlate with their

susceptibility to chemical induction of lung tumors (164). We have recently performed a

lung tumor bioassay in heterozygous Kras2-deficient mice to evaluate the effect of presence of the wild type Kras2 allele on lung tumorigenesis (165). Mice with a

heterozygous Kras2-deficiency had an increased susceptibility to the chemical induction

of lung tumors when compared to wild type mice (165), suggesting a tumor suppressor

role for the wide type Kras2 gene. Futhermore, Kras2+/- mice retaining an A/J Kras2

allele are more susceptible to lung tumor progression than those retaining C57BL/6J

Kras2 allele, as reflected in tumor size difference (165). However, treatment of mice

61 heterozygous for Kras2-deficiency produced four times as many tumors per lung as

Kras2 wild type mice regardless of the remaining allele being wt A/J Kras2 allele or wt

C57BL/6J Kras2 allele (165). This result suggests the Kras2 gene is not likely to be the major candidate for Pas1 QTL in term of its effect on lung tumor multiplicity, since the

Pas1 locus has been reported to be responsible for more than 20-fold difference in lung tumor multiplicity between A/J and C57BL/6J mice (118-120, 157, 158).

Thus, it is possible that another gene(s) exists in the Pas1 locus responsible for tumor multiplicity between the susceptible and resistant strains of mice. In Chapter 4, we performed candidate gene screening in the minimum candidate region and identified several Pas1 candidate genes (Pas1c).

62 D6Mit54 Pas1 D6Mit373 A/J C57BL/6J D6Mit54 D6Mit373

Chr.6 26.1cM /~36Mb F1 C57BL/6J

N2 D6Mit54 Pas1 D6Mit373 C57BL/6J continually marker –assisted backcrossing

D6Mit54 N7 Pas1 D6Mit373

alleletype N8 congenic determination substrain three C57BL/6J female mice N9 congenic substrain Data analyses

Urethane- induced lung tumorigenesis

Figure 3.1 Flowchart of fine mapping Pas1 locus using congenic strategy.

63

cM

Chr.6

D6Mit54 48.2 D6Mit10 48.7

D6Mit52 61.4 D6Mit61 62.3 D6mit13 63.6 D6Mit59 67.0

D6Mit57 71.1 Kras2 71.2 V D6Mit14 71.3 N

D6Mit15 74.0 D6Mit37 74.3

3 5 7 9 11 LOD Score

Figure 3.2 Genetic linkage map and the Pas1 QTL on mouse chromosome 6. The

LOD score curves for the linkages of tumor number (N, solid line) and total tumor volume (V, dotted line) are shown on the right of the chromosome. The region between

D6Mit54 and D6Mit373 was selected during the congenic backcross. The map position is based on Mouse Genome Database (MGD).

64

MCO11 MCO10

373 1557 59 52 54 Lung tumor susceptibility

QTL 3 NV(mm)

Congenic Strain (AB) 6.0 1.91 1 5.7 1.84

2 6.1 3.05

3 5.2 2.05

Subcongenic 4 1.1 0.20 65 strains 5 5.8 2.30

6 1.5 0.18

7 0.9 0.36

8 1.0 0.14 Control Strain (BB) 0.7 0.18

RECIPIENT (C57BL/6J) STRAIN DONOR (A/J) STRAIN

Figure 3.3 Fine mapping of Pas1 QTL using congenic mice. Thin line represents genomic component of C57BL/6J, and dark solid bar represents genomic components from A/J. The “D6Mit” was ommitted from each marker name. Detail statistic informtation for each strain has been shown in Table 3.1.

65

Tumor P value Total Tumor P value Animal Strain Number (t-test) Volume (t-test) Number Congenic strain (AB) 6.0±2.3 0.0000092a 1.91±0.99 0.001723 8 SS 1-741c 5.7±1.5 0.0000002a 1.84±0.73 0.001233 7

SS 2-76 6.1±2.9 0.001229a 3.05±1.99 0.004457 7

SS 3-250 5.2±1.5 0.000603a 2.05±1.18 0.005688 5

SS 4-755 1.1±1.2 0.000126b 0.20±0.29 0.001549 8

SS 5-133 5.8±2.5 0.001582a 2.30±1.40 0.006833 6

SS 6-18 1.5±1.2 0.000236b 0.18±0.22 0.001594 8

SS 7-766 0.9±0.6 0.000157b 0.36±0.24 0.002682 8

SS 8-761 1.0±0.8 0.000127b 0.14±0.14 0.001498 7

Control Strain (BB) 0.7±0.8 0.18±0.14 8

Table 3.1 Pas1 QTL effect on both lung tumor multiplicity and size a.Compared with control strain BB. b.Compared with congenic strain AB c.Subcongenic strains (SS) shown in Figure 3.3.

66 Marker Origin Method Primer Sequences (5’→3’) F: CTATGGCTGGGACTCCAAATC Pik3c2g SNP 3’UTR of Pik3c2g PCR-SSCP R: AGTCTGACATTCTGAGTTTGG F: CAATGGAGAGTGTTGGAACGG Cappa3 SNP Coding region of Cappa3 PCR-SSCP R: CTCCAAGCTCTCCTTTAGGTC F: GCTCTGATCAAGTCTGCAG Sox5 Intron of Sox5 PCR R: TAAGGCTGAAACCTCATCC F: GATGGTGATTAGAGTCAACAT D6Osu1 Celera mouse genome PCR R: CCACTCTAGAGGGTAATTGTG F: TCTTTCTCCACATGCTCATG D6Osu2 Celera mouse genome PCR R:CAGAGATGCCACAAGATGG F: TTGTCATCTGCATTCCACAG D6Osu3 Celera mouse genome PCR R: ATCTTAAGTCTGCTGTCTGC D6Osu4 F: ATCCAGCAACAGGCAATGC Celera mouse genome PCR R: CTCTTCCAGCAGACAATG F: TGCTTCTTCACCAAATGCAC D6Osu5 Celera mouse genome PCR R:CTGGCTCTATCATTTCTCTTC F:GCATTGTTCTCAGTGTTGTTC D6Osu6 Celera mouse genome PCR R:GTGTGTGCATGCACATGTG 2-bp deletion, promotor F: GACGCTTTCAGATTTGTCCTC Bcat1 PCR region of Bcat1 R: AAGCGCGGAAGATGGAGAG F: TGTGAATGAATGCACATGCAC Lrmp SNP Coding region of Lrmp PCR –SSCP R: ATATCTTCAACACGCTCGGC deletion, 2nd intron of F: CAAGGCTTTAAACTGGGTGT D6Mco10 PCR Kras2 R: ATGCAAATACAAAGCACGGA F: AAGACTCCATCCTTGTGGTC D6Mco11 Same BAC with Kras2 PCR R: AGTACACCGTTGCTGTCTTC F: GAGAGATCATCTGAGAGTTAG D6Osu7 Celera mouse genome PCR R:CCACATCCATCATATTACAGTG F: CTCAGGTGTGGGCAATTG D6Osu8 Celera mouse genome PCR R: GCCTATTGTGGCTTAGTG F: CCCACTTTCTGCTCTCTTATC D6Osu9 Celera mouse genome PCR R: GCATAGCTATGTGCACAAGC F: AACGAGAAAGCGAGCTGTC D6Osu10 Celera mouse genome PCR R: CGTGTGTGTATGTATGTATGC F: CTCTTTGTGAGGCTTGTCAAC AK016641 SNP AK016641 PCR-SSCP R: TACTTACTGTCACCGTGGAG F: ACTGTGGTTGGCACTGTTC D6Osu11 Celera mouse genome PCR R: TCCATTAAGTCAGGGCAGTC 9-bp deletion, intron of F: AGACAGGTCTGGCTGAATTC Itpr2 PCR Itpr2 R: TGAACTCAGAGATCCACCTG

Table 3.2 New polymorphic markers between D6Mit59 and D6Mit15 67 Celera Mouse Genome Location Celera Gene ID Known Gene Symbol Gene Description

145,055,086-145,115,548 mCG13310 Eca39/Bcat1 Branched chain aminotransferase 1, cytosolic 145,189,964-145,249,134 mCG13301 Lrmp/Jaw1 Lymphoid-restricted membrane protein 145,248,888-145,265,822 mCG13308 145,285,425-145,290,824 mCG13311 AK015530 Growth Hormone -inducible soluble protein 145,290,331-145,324,539 mCG13312 Kras2 Kirsten rat sarcoma oncogene 2 145,331,818-145,332,098 mCG1027108 145,340,897-145,341,275 mCG1027072 Similar to 40S ribosomal protein S25 145,377,523-145,383,294 mCG1027183 145,388,589-145,390,646 mCG1027184 145,391,854-145,392,891 mCG115945 Lactate dehydrogenase psudogene 145,403,203-145,415,908 mCG1027185 145,442,171-145,442,530 mCG1027186 145,460,150-145,460,954 mCG13304 60S ribosomal protein 145,465,863-145,503,064 mCG13305 Ak016641 Intermediate filament-related, alternatively splicing 145,522,764-145,553,102 mCG1027187 68 145,567,794-145,612,742 mCG1027188 145,617,456-145,689,483 mCG13307 145,691,244-145,696,651 mCG13302 Tuba7 Tubulin alpha 7 145,728,822-145,729,551 mCG1027189 145,746,743-145,748,292 mCG1027109 145,765,299-145,774,171 mCG1027190 145,824,887-145,889,776 mCG13306 Coiled coil protein 145,933,401-145,934,795 mCG116148 145,934,951-145,937,555 mCG15022 Bhlhb2l-pending Basic helix-loop-helix domain containing, class B

Table 3.3 Genes located in the minimum Pas1 candidate region

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CHAPTER 4

CANDIDATE GENE

SCREENIING FOR PULMONARY ADENOMA

SUSCPETIBILITY 1(PAS1) QTL

4.1 Introduction

Inbred strains of mice exhibit various susceptibilities to spontaneous and chemically induced lung tumor development (156). Results from genetic studies have

revealed that the mouse lung tumor susceptibility is governed by multiple quantitative

trait loci (QTLs) in mouse genome (114). One of these QTLs, Pulmonary adenoma

susceptibility 1(Pas1), has been mapped to mouse distal chromosome 6 in different

genetic analyses (118-120, 157, 158).

Although the Pas1 is a major determinant of mouse lung tumor susceptibility, the

gene(s) for the Pas1 QTL has not been identified yet. One of challenges was to generate a

sufficiently small candidate region for Pas1 candidate gene screening. To date, several

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strategies have been utilized to fine map the Pas1 QTL. Using physical mapping and

linkage disequilibrium (LD) strategy, Manenti et al. has fine mapped the Pas1 locus into

a ~1.5-Mb candidate region on mouse chromosome 6 (146). Independently, two fine

mapping strategies have been utilized in our laboratory to fine map the Pas1 QTL.

Linkage analysis on the F11 generation of Advanced Intercross Line (AIL) mouse

population has resulted in a ~1.3-Mb Pas1 candidate region flanked by the markers

D6Osu6 and D6Mit294 (see Chapter 2). Furthermore, a less than 1-Mb minimum

candidate region encompassed by the markers D6Osu6 and D6Osu11 has been generated

with congenic strategy (see Chapter 3).

Mouse genome maps generated by Celera Genomics and MGSC (Mouse Genome

Sequencing Consortium) are valuable resources for gene identification with the positional

candidate approach. Based on the map information, we have performed candidate gene

screening for the Pas1 QTL in our minimum candidate region and identified several Pas1

candidates (termed as “ Pas1c” genes) for future functional analyses.

4.2 Materials and methods

DNA and RNA preparation

Inbred mouse strains were purchased from Jackson Laboratory (Bar Harbor, ME).

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DNA was isolated from tail snips as described above. For RNA isolation, 100 mg of lung tissue was pulverized and total RNA extracted using TRIzol reagent according to the manufacturer’s protocol (Life Technologies, Gaithersburg, MD). The quality of the

isolated RNA was assessed by absorbance at 260 nm, the A260/A280 ratio, and

electrophoresis on 1% agarose/formaldehyde gels that indicated the intensity and

integrity of the 28S and 18S bands. Two microgram of total RNA was used in a reverse-

transcription reaction to synthesize the first strand cDNA using oligo-dT primer.

Sequence for mouse HOJ-1 homolog (mHoj-1)

We derived the mRNA sequence of mHoj-1 by blasting its human homolog HOJ-

1 (GI: 4009349) against the RIKEN and Celera mouse genome databases. The mouse

mRNA sequence was confirmed by RT-PCR analysis and has been deposited in

Genebank (AF424737, GI: 23338217).

Exon prediction and 5’-RACE assay for Pas1c2 gene

The main coding region sequence of Pas1c2 gene was independently predicted

using five exon-prediction softwares: GENESCAN, GRAIL, Genefinder, xpound and

Fgene. Multiple sets of primers designed based on the consensus coding sequence were

used in RT-PCR for confirmation. 5’- RACE assay was performed to generate full-length open reading frame (ORF) of the gene. The SMART RACE cDNA amplification kit

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(Clontech Laboratories, Palo Alto, CA) was used following the protocol provided by the

manufacturer. Briefly, RACE-ready cDNA was first prepared from mouse lung tissue using SMART technology. Then, gene-specific primers and 5’-end primers will be used to PCR amplify 5’-end cDNA fragment. The 5’-RACE PCR product was then subjected to direct sequencing. Full-length ORF was constructed based on sequence information.

We have derived the human Pas1c2 cDNA sequence by blasting mouse Pas1c2

cDNA sequence against Celera database (CDS 3.6). The derived sequence was validated by RT-PCR analyses.

Nucleotide polymorphism analyses

The mRNA sequences of Eca39, Lrmp, Ak015530, Ak016641/Pas1c1 and Krag

genes were retrieved from the NCBI Genebank (GI: 6680771, 6678713, 12853911,

12855487 and 437199, respectively). The coding region sequences of mHoj-1 and

Pas1c2 genes were generated in the present study.

For coding region nucleotide polymorphism analyses, primers were designed based on exon-flanking sequences. Using mouse lung DNA, PCR reactions were performed to amplify coding sequence fragments for each gene. PCR products were resolved on 1.2% ethidium bromide-stained agarose gels and purified using QIAquick gel extraction kits (Qiagen, Hilden, Germany). Automated sequencing was performed using dideoxy terminator cycle sequencing kits (Applied Biosystems) and Applied Biosystems

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model 377 DNA sequencers (Perkin-Elmer, Foster City, CA). Both directions were sequenced for each fragment to assure sequence fidelity.

Gene expression analyses

For gene expression analyses, the following specific primer sets were designed:

Eca39, forward 5’-ATG AAG GAC TGC AGT AAT GG-3’, reverse 5’-AGT TCC ACA

GCG TAG TGC-3’; Lrmp, forward 5’-AAG AGG GTG AAG CTT GAA GAG-3’, reverse 5’-TAC ATC CGC TTC AGG TTC TC-3’; Ak015530, forward 5’-GCC AAT

TCG TTA CGA GGA GA-3’, reverse 5’-TCA GAC TTT GGT ATC TGA ATA G-3’;

Ak016641/Pas1c1, forward 5’-GGA AGT AGA GAT TGG AAA CCA C-3’, reverse 5’-

CAA CTC TAC AAT AGT TAC TCA CG-3’; mHoj-1, forward 5’-TCT GAA GCC

AGA GCC ATG AC –3’, reverse 5’-CAC CTT CTG CCT GAA CTC AG-3’; Pas1c2, forward 5’-CGA AGA AGT AGT TCT GTG GC -3’, reverse 5’-GAC CAA AGC CGA

GCG ACT GCGGC-3’. For β-actin, forward 5’-TGA CAT CCG TAA AGA CCT CTA

TGC C-3, reverse 5’-AAG CAC TTG CGG TGC ACG ATG GAG-3’. The linear amplification region for each gene was pre-determined by plot experiments. The reaction profile generally consisted of one cycle at 95˚C for 2 mins, followed by 26 cycles at 94

˚C, 55 ˚C, and 72 ˚C each for 30 sec. Twenty-two cycles of amplification was used for the β-actin control. Thirty cycles of amplification was used for both the Eca39 and

Ak015530 genes; 32 cycles and 1 min extension time for the Ak016641 gene. The PCR

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products were resolved in 1.2% ethidium bromide-stained agarose gels and visualized

under UV.

Bioinformatics

Candidate regions from Celera Genomics and MGSC were compared. Gene

transcript sequences from one of these two sources were searched against the other to find overlapping genes using Basic Local Alignment Search Tool (BLASTn)(166).

Function domain predictions were performed for mHoj-1, Pas1c1 (Ak016641), Ak015530

and Pas1c2 genes using NCBI Conserved Domain Database (CDD)

(http://www.ncbi.nlm.nih.gov/Structure/cdd). The exon-intron boundaries for

Ak016641/Pas1c1 gene were produced by BLASTn gene mRNA sequence against Celera

mouse genome database (CDS3.6).

4.3 Results

High consistency shared by Celera and MGSC Pas1 candidate regions

Celera Genomics produced its mouse genome map by taking the whole-genome-

shotgun (WGS) assembly strategy. The public Mouse Genome Sequencing Consortium

(MGSC) has used a different assembly strategy, called hierarchical shotgun (HS)

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assembly, to produce its own mouse genome map. Due to the different assembly

strategies, it is possible that the assemblies from these two sources might have some

discrepancies. We have compared the latest MGSC genome map (MGSCv3) with the

latest Celera mouse genome map (CDS 3.6) for the fine-mapped Pas1 region and the result has been shown in the Table 4.1. These two maps are highly consistent in terms of the size of Pas1 region and overlapping gene number, which suggest that we can rely on these two maps to perform the Pas1 candidate gene screening.

mHoj-1 gene is a putative RasGTP effector

By searching NCBI human genome database, we found human HOJ-1 gene is located in the Pas1 human syntenic region 12p11 and its protein has been termed as

“human carcinoma associated” by Hoon and Yuzuki (GI: 4009349), although no experimental evidence has been published yet. We blasted the human HOJ-1 mRNA

sequence against the RIKEN mouse full-length cDNA database and Celera mouse

genome database and a complete open reading frame (ORF) sequence of the mouse

homolog (mHoj-1) was generated. The mHoj-1 gene has a 1260 bp ORF encoding a

putative 419 amino-acid protein and the translation start codon ATG is within a typical

Kozak sequence (ACCATGG). Furthermore, we performed RT-PCR using primers

derived from the mHoj-1 transcript sequence and the whole ORF was successfully

amplified. Searching the NCBI Conserved Domain Database (CDD) using the mHoj-1

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protein sequence, we found the mHoj-1 protein contain a Ras association (RalGDS/AF-6) domain at its amino terminus, suggesting it potentially function as a RasGTP effector.

The gene has been deposited into Genebank (GI: 23338217) and was later found corresponding to the Celera predicted gene mCG13306.

Pas1c2 gene is a novel gene adjacent to Kras2

The first version of mouse genome map produced by Celera Genomic in 2001 was a sequence draft and not fully annotated. We performed exon prediction for a ~200-

kb Celera genomic sequence around the Kras2 gene, which had been localized to Celera chromosome 6 through BLASTn search. The Lrmp and Kras2 genes were identified by

the programes as we expected. Surprisingly, prediction results revealed another gene

located between the Lrmp and Kras2 genes. We designed multiple primer sets and perform 5’-RACE to generate the full-length coding sequence. It turned out the gene possesses a 2193 bp ORF and encodes a 730 amino-acid protein (Figure 4.1). We have also derived the human cDNA sequence. The mouse predicted protein is 67% identities and 81% positives to the predicted human protein (Figure 4.2). In addition, it is also homologous to a Ciona intestinalis protein axonemal p83.9 (GI: 20086393, 33% identities and 52% positives, Figure 4.2). Most importantly, the gene carries a functional nucleotide polymorphism that has exhibited the tightest segregation with the Pas1 allele

in inbred mouse strains (see below). Since it is a potential Pas1 candidate, we name it as

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the Pas1c2 gene (Pas1c1 being assigned for Ak016641). The Pas1c2 corresponds to the

Celera gene mCG13308, which was annotated later by Celera and only possesses a partial

transcript sequence.

Coding region nucleotide polymorphism analyses

Based on Celera mouse genome map (CDS 3.6), there are 24 known or predicted

genes in the minimum Pas1 candidate region (See Table3.3, Chapter 3). After initial screening using RT-PCR, oligonucleotide arrays (167) and computer-assisted single nucleotide polymorphism (SNP) analysis (168), we focused on six most likely candidates

(Lrmp, Pas1c2, RIKEN Ak016641, RIKEN Ak015530, Eca39, mHoj-1) for further analyses because of their relevant functions in tumorigenesis or allelic changes between

A/J and C57BL/6J mice. The coding regions of the Lrmp gene in A/J and C57BL/6J mice were first amplified by RT-PCR and directly sequenced. By comparing the sequences from these two distinct strains, we found a total of eight nucleotide polymorphisms located at codons 31, 56, 58, 60, 243, 343, 438 and 537. Among them, five polymorphisms are missense polymorphisms that lead to amino-acid alterations, namely, codons 31 (GA/GC), 56 (GG/AC), 58 (TTC/G), 438 (A/GGG) and 537 (CC/TG) (Figure

4.3). At codon 31, the hydrophilic, negatively charged aspartic acid in A/J mice has changed to hydrophobic neutral glycine in C57BL/6J mice. At codon 56, glycine in A/J mice is substituted by aspartic acid in C57BL/6J mice. At codon 58, phenylalanine in A/J

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mice has been substituted by leucine in C57BL/6J mice. Codon 438 encodes a

hydrophilic, positively charged amino acid, arginine, in A/J mice and a hydrophobic,

neutral amino acid, glycine, in C57BL/6J mice. Finally, codon 537 is located on the

carboxyl terminus, which encodes for proline in A/J mice and leucine in C57BL/6J mice.

The strain distribution pattern for these amino-acid polymorphisms was established by

sequencing PCR fragments amplified from different strains of mouse DNA. We found a

high correlation between these polymorphisms and the Pas1 allele status (Table 4.2).

Strain SM/J has been shown to be resistant to lung tumor development, possibly because it carries multiple Pulmonary adenoma resistance (Par) loci (126).

Table 4.2 also lists amino acid substitutions in the Pas1c2, Ak016641 and

Ak015530 genes. For the Ak016641 gene, we found two amino-acid changing polymorphisms at codons 218 and 258 in its coding region. These polymorphisms and its mRNA transcript pattern (see below) are highly associated with the strain Pas1 status.

Because the Ak016641 gene is a strong Pas1 candidate, we re-name it the Pas1 candidate

1 gene (Pas1c1). Pas1c2 carries a nucleotide polymorphism at codon 60, leading an amino-acid change from Asp (A/J) to Ser (C57BL/6J). Strain distribution has also shown this polymorphism tightly associated with each strain Pas1 status. The open reading frame of gene Ak015530 (from bp123 to bp383) carries one polymorphism at codon 28.

A/J strain carries GGC (Gly) while C57BL/6J strain has GAC (Asp). Strain distribution pattern shows that Pas1 susceptible strains 129/SvJ and BALB/cJ exhibit GGC at this

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codon, which is similar to A/J, but SWR/J and CBA/J carry GAC at this codon as

C57BL/6J and other lung tumor resistant strains. For two other genes, Eca39, mHoj-1, no

amino-acid changing polymorphisms were detected in their coding sequences.

Gene expression analyses

Semi-quantitative RT-PCR was used to evaluate the relative expression level of each of the above six genes across multiple mouse strains (Figure 4.4). Lrmp is expressed

in mouse lung tissues without significant difference between lung tumor susceptible and resistant strains. This result is not consistent with a previous report (169), which showed by Northern blotting that Lrmp was not expressed in mouse or human lung tissues. This discrepancy is likely because of the use of RT-PCR, which is a more sensitive technique than Northern blotting. Eca39, Ak015530, mHoj-1, Pas1c2 are also expressed in similar

amounts in lung tissues in all strains of mice examined.

When amplifying its C-terminal coding region, the Pas1c1 gene has exhibited

different isoforms of transcripts in A/J and C57BL/6J strains of mice (Figure 4.4). A/J

mice carried two Pas1c1 transcripts while C57BL/6J mice carried only the large

transcript. Further sequencing disclosed that one exon (bp 894~bp1013, encoding 40

amino acids) is spliced out in the smaller transcript without changing its entire open

reading frame (Figure 4.5a). RT-PCR data on the various inbred strains revealed that the mRNA splicing pattern of each strain was highly co-segregated with Pas1 allele status

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(Figure4.4). Three nucleotide polymorphisms were identified in the skipped exon by direct sequencing; which are located in codons 258, 266 and 269 (Figure 4.5b). Codon

258 encodes for glycine in A/J mice and glutamate in C57BL/6J. Codons 266 and 269 carry silent polymorphisms. These three polymorphisms correlate closely with the mRNA alternative splicing pattern of each strain (Table 4.2).

4.3 Discussion

Among all of the genes located in the refined Pas1 candidate region, our study identified the Lrmp, Pas1c1/Ak016641 and Pas1c2 genes as strong candidates for the

Pas1 QTL. In the Celera mouse genome, these three genes are located in a 276-kb physical region. Previous studies suggested that Lrmp gene product might be involved in lymphoid development. Specifically, Lrmp protein may be involved in developmentally regulated intracellular trafficking of antigen receptors of lymphocytes (169). Lrmp protein can efficiently deliver a COOH-terminal antigenic peptide to MHC class I molecules in a TAP-independent manner (170). In the present study, we found eight nucleotide changes in the coding region of the Lrmp gene, and five of which result in amino acid variations between A/J and C57BL/6 strains of mice. The strain distribution pattern demonstrates that these polymorphisms co-segregate with mouse lung tumor

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susceptibility. Four of the five polymorphisms in codons 31, 56, 58, and 438 are localized in the cytosolic domain of the protein. Codons 31, 56 and 438 exhibit non-conservative changes, which may lead to conformational changes in the protein. Codon 537 is localized in the lumenal domain near the COOH-terminus, which is cleaved during post- translation processing (171). Whether the codon 537 substitution affects this processing is unclear. Lrmp is expressed in lung tissues of all mouse strains examined. The human 12p region containing SOX5, KRAS2 and LRMP is frequently amplified in testicular germ cell tumors (172). It would be interesting if the same were true for mouse lung tumors.

The Pas1c1 gene encodes a 413-amino acid intermediate filament tail domain- containing protein. Although there is no available functional information, our finding that the transcript splicing pattern co-segregates with mouse Pas1 allele status provides genetic evidence for Pas1c1 /Ak016641 as a candidate gene for Pas1. The nucleotide polymorphisms in the spliced exon (codons 258, 266 and 269) may play role in determination of the splice pattern. A mechanism for exon skipping caused by nonsense or missense mutations in BRCA and other genes may also apply to the Pas1c1 gene (173).

The Pas1c2 gene is a novel gene identified in this study, encoding a 730-amino acid protein that has no homology to known functional domains. This gene is likely to have homologues in multiple species, since our study has shown it even has a homologue in Ciona intestinalis. Most importantly, it carries a functional nucleotide polymorphism in its coding sequence and the polymorphism is tightly associated with strain Pas1 status.

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The Eca39 gene was isolated by a subtraction/co-expression strategy with Myc- induced tumors in transgenic mice, and demonstrated that Eca39 is a direct genetic target

for Myc regulation (174). Although previously we found a 2-bp deletion in its promoter

region (Chapter 3), RT-PCR results show that the polymorphism actually does not affect

gene expression. The Ak015530 gene encodes for an 86-amino-acid small protein with

unknown function but it is unlikely a candidate Pas1 gene because the amino-acid

polymorphism detected in its coding region does not co-segregate with mouse lung tumor

susceptibility (Table 4.2). In the present study, we also derived the mouse mHoj-1 gene

from its human homolog HOJ-1. Although the function of the mHoj-1 gene remains to be

determined, searching the NCBI Conserved Domain Database with the protein sequence

of mHoj-1 led to a prediction of a Ras association domain (RalGDS/AF-6) in its amino

terminus. This suggests that mHoj-1 may function as a RasGTP effector. Expression

results show the above-mentioned three genes (Eca39, RIKEN Ak015530, mHoj-1) are expressed in mouse lung tissue but to similar extents between susceptible and resistant strains of mice. Moreover, there was no amino-acid polymorphism in the coding regions

except for Ak015530. Overall, these results suggest that these three genes are less likely

to be candidate for Pas1.

In conclusion, our results have established the candidacy of Lrmp, Pas1c1

(Ak016641) and Pas1c2 genes for the Pas1 QTL, and these genes may be responsible for

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differences in lung tumor multiplicity between susceptible and resistant strains. Further functional analyses on these candidate genes will lead us to identify the Pas1 gene(s).

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Total gene Number of Number of Size of the number in genes genes identified Pas1 candidate the Pas1 identified in in CDS 3.6 region (Kb) region MGSCv3 map mouse map Celera Genomics mouse genome 983.8 24 20 - map (CDS 3.6) MGSC mouse genome map 940.4 16 - 14 (MGSCv3)

Table 4.1 Comparison of the mouse genome maps from the Celera Genomics and the Mouse Genome Sequencing Consortium (MGSC).

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-----tgggcgtggcctgtcgtcttgacaaccgtgagcgttcctg ctctgcagcgttcacttttccctaagcaaagttcctgcttctgtc 1 atggctcccaaatcaaaaaaggctcccagtaagaaaaagatgacc M A P K S K K A P S K K K M T 46 aaagccgagcgactgcggctgatgcaggaggaggaggagagacgc K A E R L R L M Q E E E E R R 91 ctgaaggaggaagaagaggcgcggctgaaatttgaaaaagaagaa L K E E E E A R L K F E K E E 136 caggaaaggctagaaatacagcggattgagagagagaagtggart Q E R L E I Q R I E R E K W X 181 ctgctggaaaagaaagacctagaacgaagaagccaagagcttgaa L L E K K D L E R R S Q E L E 226 gagcttgctctgctcgagggttgttttcctgaagcagagaaacag E L A L L E G C F P E A E K Q 271 aagcgggaaattcgagctctggctcagtggaagcactacacggag K R E I R A L A Q W K H Y T E 316 tgtgatgggagccccgacccttgggttgcccaggaaatgaacacg C D G S P D P W V A Q E M N T 361 ttcattagcctgtgggaagaggagaagaaccaggcctttgaacaa F I S L W E E E K N Q A F E Q 406 gtgatggagaaaagcaaactggtgctgtcgttgattgaaaaggtg V M E K S K L V L S L I E K V 451 aagttaattttactggaaactccgacatatgagctggaccacagg K L I L L E T P T Y E L D H R 496 actgtcctgcagcatcaagggtcaattctgcgcctacaagagctg T V L Q H Q G S I L R L Q E L 541 ctcagcctgaagatcaacgtggccacagaactacttcttcgacaa L S L K I N V A T E L L L R Q 586 gctagtaacttagcagatctggacactgggaatatggagaaaatc A S N L A D L D T G N M E K I 631 atcaaagatgagaatgtcaccctgtacgtgtgggcaaacctcaaa I K D E N V T L Y V W A N L K 676 aagaatccaaggcaccggagtgtgaggttctcagagacacaaatt K N P R H R S V R F S E T Q I 721 ggatttgaaatcccaaggatcctggccacgagcaatgttgctctt G F E I P R I L A T S N V A L 766 cggcttctacacacacgctatgaccacatcacacccttgttcccc R L L H T R Y D H I T P L F P 811 attgccgtcactgagcaaaatcaraaccccgtgggagcagagcaa I A V T E Q N Q N P V G A E Q 856 gtcaacgtcgaggaaagtacagaaaaggccatgactgaagaaaag V N V E E S T E K A M T E E K 901 ctctttactgaagaaaaagctgccaacgaagatgagcagcccaag L F T E E K A A N E D E Q P K 946 gctgaacaggaaagagagctcaacttggttcaagaggagaacaaa A E Q E R E L N L V Q E E N K 991 tatgaagctatagagaacactgtcttacaaaggacttccgactct Y E A I E N T V L Q R T S D S 1036 gaaggggaggattcccaaaccacccaacttgaactggagatgaag E G E D S Q T T Q L E L E M K

Figure 4.1 Pas1c2 gene Open Reading Frame (ORF) (continued)

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Figure 4.1:continued.

1081 ctgctgagtgaagcagtcttagcagcacagctgtgcctggtagag L L S E A V L A A Q L C L V E 1126 aatgtggtggaattgccagaagcctcacaagcctacaaggtggac N V V E L P E A S Q A Y K V D 1171 ttgtgccatttctctaccctgggcggcgtgtaccacctggatgtt L C H F S T L G G V Y H L D V 1216 ctggagctgccccctcagtgcaagcctgtgaagggctgggtgcta L E L P P Q C K P V K G W V L 1261 gtggagatactccaggaaggactgcagaggtttatatatcctcca V E I L Q E G L Q R F I Y P P 1306 gacaccacagaggaacctgatccagacgtcaccttcccacccata D T T E E P D P D V T F P P I 1351 gaggtcacactggagatccacaagagcgtcatcttctttgagcgc E V T L E I H K S V I F F E R 1396 cctagggtcgtcaggtgggacaatgaaggtaaattctggcggtca P R V V R W D N E G K F W R S 1441 gatggcatcagcagtgtctattacaaccgagaagacaggctccta D G I S S V Y Y N R E D R L L 1486 accttcagtatggatactttgggccctgtgaccttgattcaggat T F S M D T L G P V T L I Q D 1531 gctcacgtgaacatgccttaccagtcctgggagatgagtccctgt A H V N M P Y Q S W E M S P C 1576 ggcatgaacaaagtccttctaatagtgaagacggttttcatggag G M N K V L L I V K T V F M E 1621 ctccagatatacatcaaggaaaacctctgcatgctggcttcagtg L Q I Y I K E N L C M L A S V 1666 aaactgaggggcaagggactcgagtttcatctaaaaggaaaatgg K L R G K G L E F H L K G K W 1711 atggctcctatacccttcattctggctttgaaagaggccgggctg M A P I P F I L A L K E A G L 1756 aacatcttccctgctgtatactcccatttttatgtggtcatcaac N I F P A V Y S H F Y V V I N 1801 aataaggtaccccaggtggagttgaaggcctatcggcaaatggcc N K V P Q V E L K A Y R Q M A 1846 ctgctgagctctgccttctcgtttggctggagcaagtggaacatg L L S S A F S F G W S K W N M 1891 gtctgcaattccacaagggttgtgattcgggtgagggaacaactg V C N S T R V V I R V R E Q L 1936 tcagaagaaacagagcaccatacctggtcgctcctcatgttcagt S E E T E H H T W S L L M F S 1981 ggtgacagagcgcagatgctcaagatgcaggaagagaacgacaag G D R A Q M L K M Q E E N D K 2026 ttctcggaggccctcagggagggcaccgagttccactccaccttg F S E A L R E G T E F H S T L 2071 taccacatgatgaaggacttcgcctcccccgtggcaatggagagg Y H M M K D F A S P V A M E R 2116 gtcaggcattcgaactgccagttcatcgactcagtgtgctacatg V R H S N C Q F I D S V C Y M 2161 ctgctgtctatccgcgtcctcagctattcctag 2193------L L S I R V L S Y S *

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Mouse MAPKSK KAPSKKKM TKAERLR L M QEEEERRLKEEEEARLK Human MS GSKKKKV TKAERLK L L QEEEERRLKEEEEARLK Axonemal p 83.9 MPPKSPNRSGK S T PTRGRPGEKKDEEKLL QDEEEE RLR

FEKEEQ ERLEIQRIER EKWxLLEK KDLERRS QELEELA LLEG CFPEAEKQ YEKEEM ERLEIQRIEKEKWH RLEA KDLERRNEELEELY LLER CFPEAEKL LE Q EEKAR Q E KEAREKLEQER RAELD TKKDKQ VFE TNIEL GAVKLE V E QV

K R E IRALAQWKHYTECDGSPDPW VAQEMNTFISLWE E EKN QAFEQ V M EKS K Q E TKLL S QWKHYIQCDGSPDPS VAQEMNTFISLWK EKT N ETFEE V I EKS K NDKLA H A E W NRY MKCDGK PDPTSVKE I NTFISLSHEKGSPDVNIV L E DA

KLVLSLIEKV K L ILLETPTYELDHRTV L Q H Q G SILR LQELLS LKI NVATE K V VLN LIEKLKF ILLETPPCDL QDK NIIQYQE SILQ LQELLH LKFGVATX KLI LSLISDL NELLEDFTP E E FEQK - V DSY RQTILS LQD LLLNRYN E ATL

L LLR QASN LADLDT GNMEKI IKDENVTLYVWANLKKNPRHRSVRFSETQI I LLKQAST LADLDSGNMEKVIKDENVTLYVWANLKKNPRHRSVRFSETQI KMLKE ASYEADSESGNLQKVVDGENE T IMLWANLN KNPRFKLFEF ENEKI

GFEIPRILATSNVA L RLLHTR YDHITPL F P IAVTEQNQNPVGAE Q V NVEE GFEIPRILATSDIAVRLLHTH YDHV S A L H P VSTP S K EYTSAVTE L V KDDV S FEL P KVLAMADIAVRI L R T KFDHY S HQCTTFLP K K KKVKDEEPIPEEPP

STEKAMTEEKLFTE EKAANE D E QPKAEQERELNLVQEENKYEAIE NTVL Q K N------VE K A ISKEVEEESKQQERGSHL I QEEEIKVEEE QGDIE K P------E D A EEVEVKGDEENGE DAKSVV E E GRQSKQSNEPGL V

RTSDSEG E D S QTTQLE L EMKL LSEA V L AAQLC LVENVVE L PEASQAYKVD VKMSS A EEE S EAIKCE R EMKV LSET V S AAQLL LVENSSE K P DFFEDNVVD NEGEKEEETKKD-----E NEGEKE DAVKTPDVQIE IEDDEEE ILDPDVVD

LCH FSTLGGVYHLDV LELPPQCKPVKGWV LVEILQ EGLQRFIYPPDTT-- LCQFT TLGGVYHLDI LELPPQCKPVKGWMIVEILK EGLQKYTYPPE TT-- L R QFSP LGGVYHVDL L KTPPQPNIV R GWT L TQI IDKPLSTVKYPS D NPNT

------EEPDPDVTFPPIEVTLEI H KSVIFFER P R V ------EEFETE NAFPPIEVTLEV H E NVIFFED PVV GRSSSRVASANPEGRDEGSPSKTPLE QQQPPIGLT FALPSNVM FFEE P Q V

VRWDN EGKF WRS DGISS V Y Y NRE D RLLTFSM DTL GPVTLIQDAHVNMPYQ VRWDN EGKF WRS DGISS V Y Y NRE D RLLTFSM DTL GPVTLIQDAHVNMPYQ ASWDSSDKHWK T S GITDTNFDEE N R K L L F KTQEFGTFCL M QDSHL NMPF Q

Figure 4.2 Pas1c2 protein homology among mouse, human and Ciona

intestinalis (Continued). 87

Figure 4.2: continued

SWEM SPC GMNKVLLIVK TVFM ELQIY IKENLCMLASVKLR GKGLE FHL K G SWELR PLDVNKVLLTVTTVFT E I QIQ IKENLCMLS S I KLKD KKHIS I L E G SWELK P K G T N STVLTI T AAIAE VEI EVK DSKC R L N A PAEDPPK ELS G L Y G

KWMAPIPFILALKEAGLNIFPAVYSHFYVVINNKVPQVELKAYRQMALLS T WMTPIPFII ALKEAGLNIFPTRHSHFYVI INNKVPL VEV KAYRQMALLS KWMAVPKLI A A MRDAGV N V FPAEDSHKFV S I QSK EVDLE R-VY E QMAI LS

SAFSFGWSKWNMVCNSTRVVIRVR-----E Q LSEE- TEHHTWSLLMFSG SAFAFGWSKWNLLCNSTK VVFK VR-----EHLTEEC TENPNWALLMFSG S T FAFS WSKWNNDAGS KQV IIQIAPCLIKE NVPRDAVSDDDWSIFSVS D

DRAQMLKMQEEN D K FSEALREGTEFHSTLYHMMKDFASPV AMERVRHSN DRAQR LKIKEESEA FSEALK E E TEFHSTLYHMV KDFASE EAMEKVRS SN D MSYKL ALSE YDE E F ADVVAKG ATYH CDL L H AQYERQPLKTATK NCWNN

CQFIDSVCYMLLSI RVLSYS -730730 CQFVNSVCH MLLSTRLLSYS 716 SPKNHKTRTSFSFTRLPHY 737

Figure 4.2 Pas1c2 protein homology among mouse, human and Ciona intestinalis. Residues identical in at least two species are shaded in black. In mouse protein, the codon 60 (“x”) encodes an Asparagine (AAT) in A/J mice and a Serine

(AGT) in C57BL/6J mice. The human protein sequence (67% identities and 81% positives) is based on our predicted human Pas1c2 cDNA sequence. The mouse

Pas1c2 protein is also homologous to a Ciona intestinalis protein axonemal p83.9

(GI: 20086393, 33% identities and 52% positives).

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Figure 4.3 Nucleotide polymorphisms in the Lrmp gene that alter amino acids. a. distribution of amino-acid changing nucleotide polymorphisms in Lrmp coding region.

Only polymorphism-bearing exons are shown. Dashed line represents intronic region; solid line, exons. b. chromatograms of the amino-acid changing nucleotide polymorphisms.

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a. 369446 516 517 598 599 645 1714 1821 1822 2313

ATG

ACG GAA GAC TCC GAG AGG ACC AGG ACG GAA GGC TCC GAG GGG ACC AGG

GCT GGC CAC TTC TAT CCA CCG CCA GTG TGA GCT GAC CAC TTG TAT CCA CTG CCA GTG TGA

Codon 31 GAC Codon 56 GGC Codon 438 AGG Codon 537 CCG b. Codon 58 TTC

A /J

Codon 31 GGC Codon 56 GAC Codon 438 GGG Codon 537 CTG Codon 58 TTG

C57BL/6J

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Pas1 Pas1c2 Lrmp Ak015530 Pas1c1(Ak016641) Inbred

strains alleletype Codon 60 Codon31 Codon56 Codon58 Codon438 Codon537 Codon28 Codon218 Codon258

A/J Pas1/s AAT GAC GGC TTC AGG CCG GGC CGC GGA 129/SvJ Pas1/s AAT GAC GGC TTC AGG CCG GGC CGC GGA BALB/cJ Pas1/s AAT GAC GGC TTC AGG CCG GGC CGC GGA SWR/J Pas1/s AAT GGC GGC TTC AGG CCG GAC CGC GGA

CBA/J Pas1/s AAT GGC GGC TTC AGG CCG GAC CGC GGA

91 SM/J Pas1/s AAT GAC GGC TTC AGG CCG GGC CGC GGA C57BL/6J Pas1/r AGT GGC GAC TTG GGG CTG GAC CAC GAA SJL/J Pas1/r AGT GGC GAC TTG GGG CTG GAC CAC GAA AKR/J Pas1/r AGT GGC GAC TTG GGG CTG GAC CAC GAA C3H/HeJ Pas1/r AGT GGC GAC TTG GGG CTG GAC CAC GAA DBA/2J Pas1/r AGT GGC GAC TTG GGG CTG GAC CAC GAA

Mus. Spretus Pas1/r AGT GGC GGC TTG AGG CCG GAC CGC GAA

Table 4.2 AA-changing nucleotide polymorphisms in the Pas1c2, Lrmp, Ak015530 and Pas1c1 (Ak016641) genes

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Figure 4.4 RT-PCR expression pattern of six genes. Semi-quantitative RT-PCR results for Pas1 candidates in lung tumor susceptible and resistant strains of mice are shown.

Primer information is described in Methods. DNA ladder (100 bp) was used in agarose gel electrophoresis.

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M A/J DBA/2J SWR/J SM/J BALB/cJ AKR/J Mus.Spre. SJL/J C3H/HeJ CBA/J 129/SvJ C57BL/6J

Pas1c1 (Ak016641)

mHoj-1 600bp

Eca39 93 Lrmp

Ak015530

Pas1c2

β-actin

93

a

180 259 260 510 511 706 707 893 894 1013101412191220 1392 1393 1534

TAA ATG (1421bp)

b 881 C TCC ACG GTG ACA GTG TGG GCA GCA GCT TCG

GAA GCA AAG CCA CAG CCA CCA ACG GAC TTT GTT TGG GAG GG/AA CAG AGC AAG TTC CGA TCC AGT CCA/G GAC TGC ACA/G ACC ATC TTG TGC AAG CCC AAT GGT GAG

GCC ATT GCC TGG TAC ACT CCT ATC CAC 1040

Figure 4.5 Alternative splicing of Ak016641 mRNA. a. One exon encoding for 40 amino acids is spliced out in A/J and other Pas1/s strains, leading to two transcripts in

Pas1/s strains and only the longer transcript in Pas1/r strains: solid represents the shorter transcript and hatched represents the longer transcript. b. Nucleic acid sequence in the skipped exon. The three polymorphic codons are in boldface; each of these co-segregated with specific Ak016641 isoforms.

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CHAPTER 5

FINE MAPPING AND CANDIDATE

GENES ANALYSES FOR MOUSE PULMONARY

ADENOMA RESISTANCE 2 (PAR2) QTL

5.1 Introduction

Lung cancer is the leading cause of cancer-related death in the United States (1).

Despite advances in diagnosis and treatment of the disease have been made in past two decades, the prognosis of lung cancer is still dismal with the overall 5-year survival rate less than 15% in the United States (8). Although cigarette smoke is implicated in about

90% of male and 75-80% of female lung cancer deaths (86), only about 10% of heavy smokers will ultimately develop lung cancer (87), which suggests that there is a variation in individual susceptibility to lung cancer. Indeed, segregation analysis has shown susceptibility to lung cancer may be inherited in a Mendelian fashion and a rare autosomal dominant cancer-causing gene may play a role (95).

Investigation into the inheritance of human lung cancer, however, requires the 95

identification and evaluation of families with many lung cancer-affected individuals. This has been hindered by the poor lung cancer prognosis. Besides, prevalence of environmental factors that contribute to the lung cancer development can also confound genetic effects (175). Other factors may also negatively affect this course (see Chapter 1).

On the other hand, inbred strains of mice vary markedly in their susceptibility to spontaneous and chemical-induced lung tumors (156), thus representing a valuable model for us to study lung cancer genetics. Based on their mean tumor multiplicities induced by lung carcinogens such as urethane, the inbred mouse strains can been categorized into sensitive, intermediate and resistant groups (114). The A strain is the most susceptible strain while the C57BL/6 strain is the most resistant. Other strains like the BALB/c strain and the 129 strain belong to the intermediate group and are less susceptible to lung tumorigenesis than the A strain (114).

Experimental crosses of inbred mouse strains have revealed that there exist both

Pulmonary adenoma susceptibility (Pas) loci and Pulmonary adenoma resistance (Par) loci in the mouse genome. Studies on recombinant inbred (RI) strains, F2 hybrids and backcrosses between A/J and C57BL/6 inbred strains have suggested that there are at least four Pas quantitative trait loci (QTLs) associated with urethane-induced pulmonary adenomas in mice (116, 118-120, 157, 158). Using recombinant congenic (RC) strains, even more mouse lung cancer susceptibility (Slucs) loci have been mapped and complex interactions between these genes have been found (127-130).Among all of these Pas loci,

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the Pulmonary adenoma susceptibility 1(Pas1) play a major role and has been mapped to the mouse distal chromosome 6 by using (A/J × C3H/HeJ) F2 mice (102-104, 136, 137).

In addition to Pas loci, several Pulmonary adenoma resistance (Par) loci have also been identified (121-123, 126). Inbred mouse strains A/J and BALB/c carry the same

Pas1/+ allele but show different susceptibility to the urethane-induced lung tumors. In

(A/J x BALB/c) F1 mice, the relatively resistant BALB/c phenotype was dominant over the high susceptibility of A/J mice (176). Further analysis on F2 hybrids and backcross mice supported the hypothesis that a single gene accounts for the difference in adenoma susceptibility between A/J mice and BALB/c mice (176), although a later study suggested that at least four loci and sex were associated with susceptibility to the chemical induction of lung adenomas in (A/J x BALB/c) mice (124). A major resistance locus, designated as Pulmonary adenoma resistance 2 (Par2), has been mapped to the mouse chromosome 18 independently by several groups and accounts for 15% ~40% phenotype variance (122-124).

The use of congenic mice to fine-map quantitative trait loci (QTLs) has been used frequently to facilitate positional cloning of candidate genes. One objective of this study was to utilize congenic and subcongenic mice to narrow down the Par2 candidate region.

Previous studies have identified the region potentially harboring the Par2 QTL (122-

124). In the present study, an approximately 28-cM region on distal chromosome 18 encompassed by the D18Mit34 and D18Mit4 markers was selected and multiple congenic

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and subcongenic strains containing various Par2 QTL regions were generated. The overlapping QTL-containing regions resulted in a 6.3-Mb candidate region flanked by the marker D18Mit103 and the marker D18Mit162. By constructing a mouse/human comparative map, a high homology between the mouse Par2 candidate region and its human syntenic region on chromosome 18q21 was observed. Real-time RT-PCR analyses have suggested that four genes are possible Par2 candidates based on their

differential expression level, including Myo5b, Smad7, Mapk4, and GABA-A receptor-

like gene mCG58197. More interestingly, sequencing analyses for this region found that

the Rad30b gene, encoding the DNA–dependent polymerase iota (Polι), carries 25

nucleotide polymorphisms in its coding region between A/J and BALB/c inbred strains,

causing ten amino-acid alterations. Functional analyses on the above five genes are

needed to clarify their Par2 candidacy. In addition, 54 putative genes exhibited various types of SNPs when comparing genomic sequences from A/J, 129X1/svJ, 129S1/svImJ,

DBA/2J, and C57BL/6J mice. Screening of these SNPs in the future may help us find polymorphic markers and identify new Par2 candidate genes.

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5.2 Materials and methods

Construction, genotyping and phenotyping of congenic/subcongenic mice

The congenic mice used in the present study were constructed using inbred strains

A/J and BALB/c purchased from the Jackson Laboratory (Bar Harbor, ME). The breeding design in the study was to put an approximately 28-cM fragment of chromosome 18, encompassed by D18Mit34 and D18Mit4 markers from lung tumor susceptible A/J strain, onto the genetic background of lung tumor more resistant BALB/ c mice. Selection of this chromosome region governed the selection of mice for mating until N7 generation. A/J mice were initially crossed to BALB/ c mice. F1 progeny were backcrossed to BALB/ c mice to produce the first backcross generation (N2). N2 heterozygous for the chromosome region of interest were then backcrossed with BALB/c mice to produce the N3 generation. At N5, markers for other Par QTLs were genotyped to assure a clean background. At N8, 81 male subcongnic strains containing different chromosomal regions of interest were generated. These individual strains were then each crossed to three BALB/ c females to produce the N9 generation. An average of 4 to 6 N9 subcongenic mice were generated from each N8 subcongenic strain. The following markers were used to determine Par2 region genotypes: D18Mit34, D18Mit119,

D18Mit202, D18Mit36, D18Mit122, D18Mit207, D18Mit103, and D18Mit162,

D18Mit155, D18Mit4. For DNA isolation, tail clippings from each mouse were

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homogenized and incubated overnight at 37°C in nucleic acid lysis solution (pronase 0.4 mg/mL, 10% sodium dodecylsulfate [w/v], 10 mM Tris, 400 mM NaCl, and 2 mM

EDTA) followed by saturated NaCl extraction, precipitation with ice-cold 100% alcohol, and washing twice with 70% alcohol. All of the mouse microsatellite primers were purchased from Research Genetics (Huntsville, AL). The forward primer was end-labeled with γ-32P-ATP, and 30 cycles of polymerase chain reaction (PCR) were performed at

94°C for denaturation, 55°C for annealing, and 72°C for extension. 8% denaturing polyacrylamide gels were used for resolution of the radioactive-labeled PCR products followed by autoradiography. For assessing lung tumorigenesis in these congenic mice,

5-week-old N9 mice were given a single intraperitoneal (i.p) injection of urethane (1 mg/g body weight) in 0.2 mL phosphate buffered saline (PBS) .All animals were euthanized by CO2 asphyxiation 7.5 months after urethane initiation. The lung tumor number for each mouse was counted using a dissecting microscope.

Construction of mouse/ human Par2-region comparative map

The comparative map was constructed based on information derived from Celera

CDS3.6 databases (Celera Genomics, Rockville, MD). The D18Mit103 marker is at

68.5Mb on the Celera mouse genome map. The D18Mit162 marker was anchored to the map using BLASTN sequence analysis tool. The marker sequence was obtained from the

Whitehead Institute (http://www-genome.wi.mit.edu/cgi-bin/mouse). In order to define

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the human syntenic region, the hCG1658980 and DKFZP564D1378 genes were selected.

The mouse homologs of hCG1658980 and DKFZP564D1378 genes were found located near the D18Mit103 and D18Mit162 markers, respectively. Mouse transcript sequences in the Par2 candidate region were searched against the Celera human transcript database using BLASTn. The homologous relationship between mouse genes and human genes was thus established.

Real time RT- PCR assays

Total RNA was isolated using TRIzol reagent according to the protocol from the manufacturer (Life Technologies, Gaithersburg, MD). RNase-free DNase was used to avoid DNA contamination in the samples. The quality of the isolated RNA was assessed by absorbance at 260 nm, the A260/A280 ratio (1.7-1.9), and electrophoresis on 1% agarose/formaldehyde gels showing intensity and integrity of the 28S and 18S bands.

Reverse transcription reactions with Superscript RT (Life Technologies, Gaithersburg,

MD) were used to make cDNA from the total RNA, and these samples were aliquoted and stored at -80˚C for use in real time RT-PCR reactions. Real time RT-PCR was performed using a Perkin-Elmer ABI 7700 Sequence Detection System (Applied

Biosystems Inc.) and the SYBR Green DNA PCR Core Reagent Kit as recommended by the manufacturer. Each reaction was performed in a 50 µl volume with a final concentration of 4 mM MgCl2, 1X SYBR Green Buffer, 300 µM dNTPs with dUTP, 200

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µM primers, 0.025 U/µl of Amplitaq Gold, 10 µl of a 1:10 dilution of the cDNA and

RNase free water. The thermal cycling conditions for real-time RT-PCR were 10 minutes

at 95 ˚C for activating AmpliTaq Gold, followed by 40 cycles of denaturation for 15

seconds at 95 ˚C and annealing/extention for 30 seconds at 60 ˚C. β-actin mRNA was

quantified to normalize the amount of RNA in each sample. Amplified products were analyzed using 3. 5% agarose gel electrophoresis with ethidium bromide and visualized

under UV radiation. PCR primers were designed using PrimerExpress software (PE

Biosystems Inc.).

Nucleotide polymorphisms analyses

Polymerase chain reactions were performed to amplify the coding sequences

using cDNA prepared from A/J and BALB/c mouse lung tissue. Five primer sets were used to cover whole coding region of Rad30b gene. Primer set 1: F, 5’-GAG GAA GAA

GAC GCT CCT C-3’, R, 5’-TTC CTC CAA CAA TTC TGT GAC. Primer set 2: F, 5’-

GAG CCG CTA CAG AGA GAT G-3’, R, 5’-GTA GTA AGA CCG TCT GCT G.

Primer set 3: F, 5’-GTG GCT CCT AAT AAA CTC TTG-3’, R, 5’-AGG CCC TTT CTT

AGC ACT GC-3’. Primer set 4: F, 5’-ATG GTG AAC GTG AAG ATG CC-3’, R, 5’-

GCC TTG ACT CGT TTG CTC AT-3’. Primer set 5: F, 5’-TCG TGC GGA AAG GAC

TGT TC-3’, R, 5’-ACT AGA ATT TCC TTC CTG TGC-3’.

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In this study, potential candidate genes Mbd2, mCG1033415, Dcc, Smad4,

mCG22648/Mapk4, RikencDNA8430417G19/Elac1, Smad7 and Smad2 were also selected for sequencing analyses. Primers were designed based on Celera transcript sequences and will be available upon request.

Generally, the following PCR condition was used for each primer set: 95˚C for 2 min, followed by 30 cycles of 94˚C for 30 sec, 55˚C for 30 sec and 72˚C for 1 min, finally 72˚C for 6 min. Annealing temperature was modified for each primer set. After electrophoresis on 1.5% agarose gels, the PCR products were purified with QIAquick gel extraction kit (QIAGEN, Valencia, CA) and subjected to direct sequencing.

Computational Single Nucleotide Polymorphism (SNP) analyses

Single Nucleotide Polymorphisms (SNPs) in the Par2 candidate region were derived from the Celera mouse RefSNP database. The genomic sequences of A/J,

129X1/SvJ, 129S1/SvImJ, DBA/2J and C57BL/6J were used to produce the SNPs. The

6-Mb candidate region from 68.8Mb (Rad30b location) to 74.8Mb was investigated. For each gene, coding region, 5’-end and 3’-end untranslated region (UTR) were examined for potential SNPs. The SNPs in intronic region were ignored because presumably intronic sequences play less important role in gene function.

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Statistical analysis

The differences in tumor multiplicity between congenic substrains and control strain CC were evaluated by two-tail student t-tests. The difference was considered significant if the P value was less than 0.01.

5.3 Results

Congenic mapping of the Par2 locus

The region covering the Par2 QTL is approximately 28 cM encompassed by the

D18Mit34 and D18Mit4 markers. Par2 congenic strains were constructed by placing this

28-cM fragment from the A/J (donor) mice onto the genetic background of the more resistant BALB/c mice (recipient) in a total of 9 backcrosses. Nine generations of backcrosses result in congenic and subcongenic mice that are approximately 99.81 % recipient genome and carry the region of interest. In order to produce subcongenic strains, 10 male and 5 female N7 mice that carried 28 cM of the heterozygous QTL region were backcrossed to BALB/c mice. A total of 81 male subcongenic strains (N8) containing various QTL subregions were generated. Each Par2 subcongenic strain was

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crossed with three female BALB/ c mice. Urethane-induced lung tumor bioassays were carried out using the N9 generation of mice.

The congenic strain AC is heterozygous for the entire 28-cM region and the control strain CC is homozygous. A significant difference in lung tumor multiplicity was observed between the congenic strain AC and the control strain CC (Figure 5.1). The congenic strain AC had an average of 6.1±1.5 tumors/mouse, whereas the control strain

CC had an average of 2.6±1.4 tumors/mouse (P <0.0001). Thus the substitution of one

A/J Par2 allele for the BALB/c Par2 allele increased the mouse lung tumor susceptibility by 2.3-fold.

Subcongenic strains of mice contain various donor fragments (Figure 5.2).

Genotypings at the markers D18Mit34 through D18Mit4 were performed for each strain to determine the donor fragment. If the donor fragment carries the Par2 QTL, the subcongenic strain will show the high lung tumor susceptibility like the congenic strain

AC. If the Par2 A/J allele is absent in the donor fragment, the subcongenic strain carrying it will exhibit low tumor number like the control strain CC. The subcongenic strain 275 was homozygous at the D18Mit162 locus and had a tumor multiplicity of

6.0±2.4 tumors/mouse. The subcongenic strain 317 was homozygous at the D18Mit103 marker and had a tumor multiplicity of 5.5±1.7 tumors/mouse. Both strains had significantly higher tumor multiplicities than the control strain CC (P=0.0005, P=0.0033,

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respectively). Based on the congenic analyses, the Par2 QTL is now narrowed to a region

between the D18Mit103 and the D18Mit162 markers.

Comparative map between the mouse Par2 candidate region and its human syntenic

region on the chromosome18q21

In the Celera mouse genome (CDS3.6), the D18Mit103 gene is located at

68.50Mb. The D18Mit162 marker was anchored to 74.80Mb position in this study. The

physical length of the mouse Par2 region is approximately 6.3Mb and contains 79 genes.

Among of them, thirteen genes are known genes, including DNA polymerase iota

(Rad30b), methyl-CpG binding domain protein 2(Mbd2), deleted in colorectal carcinoma

(Dcc), MAD homolog 4 (Madh4/Smad4), Riken cDNA8430417G19/Elac1, mCG119144/

NAD-dependent malic enzyme (Me2), mCG22648/Mapk4, Riken cDNA2810433K01, methyl-CpG binding domain protein 1(Mbd1), myosin Vb (Myo5b), lipase (endothelial,

Lipg), MAD homolog 7(Madh7/Smad7) and MAD homolog2 (Madh2/Smad2). Another seventeen genes are unknown but their predicted protein products are similar to functional protein domains. All of the rest are functionally unknown and were predicted by computer programs.

The human syntenic region on the chromosome 18q21 is approximately 7.4 Mb in length, which is defined by the genes hCG1658980 (at 50.76Mb) and DKFZP564D1378

(at 43.32Mb). Only thirty-six genes were annotated in this human region. Most of the

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genes are known genes, including RAD30B, methyl-CpG binding domain protein2

(MBD2), deleted in colorectal carcinoma (DCC), hypothetical protein LOC51320, MAD

homolog 4(MADH 4/SMAD4), E.coli elaC homolog1 (ELAC1), NAD-dependent malic

enzyme2 (ME2), B29 gene, p63MAPK/MAPK4 gene, CpG binding protein (CGBP),

methyl-CpG binding domain protein 1(MBD1), acetyl-CoenzymeA acyltransferase

2(ACAA2), lipase (endothelial, LIPG), 60S ribosomal protein L17 (RPL17), hypothetical

protein FLJ20071, MAD homolog7(MADH7/SMAD7), KIAA0427, HEIL1, MAD

homolog2 (MADH2/SMAD2) and HSPC039 gene. In addition, seven psudogenes are

located in this region as well.

BLASTN sequence analysis tool revealed that the mCG19687 gene is the mouse

homolog of human B29 gene. The mCG22647 gene encodes the mouse CGBP protein.

The mCG22649 gene is the homolog of human ACAA2 gene. The mCG1233291 is the homolog of human RPL17 gene. The mCG119038 is homologous to human KIAA0427 gene and the mCG19147 is homologous to the human hCG1790951. Thus, there are totally twenty pairs of homologous genes found in the Par2 candidate region (Figure

5.3).

Evaluation of gene expression in the Par2 candidate region

Real time RT-PCR was used to evaluate gene expression in the Par2 candidate region. Ten genes were differentially expressed in A/J and BALB/c mouse lung tissue.

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Primer sequences and expression difference for each gene are summarized in the Table

5.1.Among of these genes, five genes, Mbd2, mCG58197, Mapk4, mCG123290 and

mCG48910, were expressed higher in BALB/c mouse lung than in A/J mouse lung. In contrast, the other five genes, Smad4, Cgbp, Mbd1, Myo5b and Smad7, were expressed higher in A/J mouse lung than in BALB/c mouse lung. Specially, the mouse mCG58197 and Mapk4 genes were expressed less than half amount in A/J mouse lung than in

BALB/c mouse lung (0.36±0.12-fold, 0.38±0.06-fold, respectively). The Myo5b and

Smad7 genes had a more than 1.5-fold expression level in A/J mouse lung than in

BALB/c mouse lung. These four genes thus were considered as the Par2 candidate genes.

Twenty-five nucleotide polymorphisms in Rad30b coding region

The mouse Rad30b gene has a 2154-bp open reading frame and encodes a 717- amino acid protein. By sequencing its entire coding region, we found total 25 nucleotide polymorphisms in Rad30b coding region between A/J and BALB/c mice, causing ten amino-acid changes (Table 5.2). At codon 4, the large, hydrophopic Leucine in A/J has been substituted by a small, hydrophilic Serine in BALB/c. At codon 7, the small, hydrophobic, neutral Glycine in A/J has been changed to a large, hydrophilic, positively charged Arginine in BALB/c. At codon 334, the neutral Glutamine has been changed to a positively charged Lysine. At codon 378, A/J has a hydrophilic Serine while BALB/c has a hydrophobic Alanine. At codon 518, A/J has a large, hydrophobic Tyrosine, and

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BALB/c has a small, hydrophilic Serine. At codon 524, the hydrophilic Asparagine has

been substituted by a hydrophobic Tyrosine in BALB/c. At codon 569, the small,

hydrophobic, neutral Alanine in A/J has been changed to the large, hydrophilic,

negatively charged Glutamic acid in BALB/c. At codon 574, A/J has a Glutamic acid

while BALB/c has an Aspartic acid. At codon 606, A/J has a neutral Glutamine while

BALB/c has a positively charged Arginine. Finally, at codon 645, a hydrophilic

Threonine in A/J has been substituted by a hydrophobic Isoleucine in BALB/c.

Single Nucleotide Polymorphism (SNP) analyses

SNP analyses in the mouse Par2 region were performed based on the Celera mouse RefSNP database (Release 1.0). These SNPs were produced based on genomic sequences of five mouse inbred strains: A/J, 129X1/SvJ, 129S1/SvImJ, DBA/2J and

C57BL/6J.The SNP searching was restricted to the 5’ and 3’-UTR and the coding region of each gene. Totally, in addition to polymorphisms detected in Rad30b gene, there are

150 SNPs located in the Par2 candidate region (Table 5.3): thirty five SNPs in 3’-UTR

region, thirteen SNPs in 5’-UTR region, fifty four mis-sense SNPs, one non-sense SNP,

and fouty seven silent SNPs. Of the total 78 genes (not including Rad30b), 54 genes

exhibited various types of SNPs.

We have sequenced the Dcc, Mbd2, mCG1033415, Smad4,

RikencDNA8430417G19/Elac1, mCG22648/Mapk4, Smad7 and Smad2 genes for

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potential SNPs. Although these genes may carry SNPs in other inbred strains as indicated in Table 5.3, so far no SNP has been found between A/J and BALB/c mice.

5.4 Discussion

In this study, we conducted an extensive congenic mapping experiment to narrow the Par2 locus in order to identify the Par2 gene. Based on previous linkage analysis studies, a 28-cM distal chromosome 18 region that harbors the Par2 locus from A/J strain

(donor) was constructed on the genetic background of BALB/ c strain (recipient) by a series of backcrosses. After nine backcrosses performed to produce congenic mice, the genetic makeup of the selected region is expected on average to be 99.81 % BALB/c genes and 0.19% residual A/J genomic background (150). We demonstrated that the Par2 locus is associated with susceptibility to mouse lung tumor development after urethane initiation in congenic mice. A semi-dominant BALB/c allele contributed approximately

2.5-fold difference in lung tumor variance between the congenic AC strain (6.1±1.5 tumor/mouse) and control CC strain (2.6±1.4 tumor/mouse). Thus, the substitution of one

A/J Par2 allele for the BALB/c Par2 allele increased the mouse lung tumor susceptibility by 2.3-fold. By carefully analyzing phenotype (i.e.tumor multiplicity) and genotype of

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each subcongenic strain, we have narrowed the Par2 QTL to a region of approximately

6.3 cM between the markers D18Mit103 and D18Mit162.

Human chromosome 18q21.1 locus is deleted in many human cancers including squamous cell carcinomas (177), osteosarcoma(178), colon cancer (179) , breast cancer

(180).Recent studies have indicated that deleted segments of 18q21 contain a tumor suppressor gene(s) that plays a role in the development of lung cancer (181, 182). Our comparative map have revealed a very conserved homology between the mouse Par2 region and its human syntenic region on chromosome18q21, suggesting that it is very likely there is also a tumor suppressor gene(s) within the mouse Par2 region affecting lung cancer development. To date, there are three known tumor suppressors (Dcc, Smad2 and Smad4) recognized in this region. However, lack of functional polymorphism or significant lung expression difference between A/J and BALB/c mice has made these tumor suppressors unlikely play the role as the Par2 gene (183, 184).

Four genes in the Par2 candidate region may be regarded as Par2 candidate genes due to their differential expression levels between the A/J and BALB/c mice. Myosins are known as actin-based motors. They play fundamental roles in many forms of eukaryotic motility such as cell crawling, cytokinesis, phagocytosis, growth cone extension, maintenance of cell shape, and organelle/particle trafficking. The human genome contains approximately 40 myosin genes that can be divided into 12 classes based on analysis of their head and tail domain structures (185). The Myo5b gene belongs to the

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class V and is expressed two-fold higher in the A/J mouse lung than in the BALB/c

mouse lung. Even though the class V myosins are one of the best characterized groups of

unconventional myosins, and myosin Va has been implicated in the regulation of vesicle

trafficking in neurons and melanocytes (186-188), much less is known of the function of

myosin Vb. One recent study using yeast two-hybrid screening system identified myosin

Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling

systems, indicating that the Myo5b gene may be involved in mediating transit out of the

plasma membrane recycling system (189).

In addition to the Smad2 and Smad4 genes, a third Smad gene, Smad7 was found located in the Par2 candidate region. SMAD proteins have been identified as signaling mediators in the TGF-β pathway, which is involved in a range of biological activities including cell growth, morphogenesis, development and immune responses (190). The activated TGF-β receptor induces phosphorylation of the Smad2 and

Smad3 proteins, which form hetero-oligomeric complexes with the Smad4/DPC4 protein that translocate to the nucleus, where they then regulate transcriptional responses. In contrast to the Smad2, Smad3 and Smad4 proteins, all of which facilitate TGF-βsignaling transduction, the Smad7 protein can be induced by TGF- stimulation but function as an

inhibitor of TGF-β signaling transduction pathway (191, 192) . A recent research

revealed that stable transfection of COLO-357 human pancreatic cancer cells with a full-

length Smad7 construct led to complete loss of the growth inhibitory response to TGF-β,

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enhanced anchorage-independent growth and accelerated growth in nude mice (193),

suggesting that the Smad7 gene may play as an oncogene.

Our results also revealed that some genes are expressed in more amounts in the

BALB/c mouse lung than in the A/J mouse lung. Mitogen-activated protein kinases

(Mapks) have been established to function as important mediators of signal transduction by growth factor receptors. Based on the real-time RT-PCR, the mouse Mapk4 gene is expressed less than half amount (0.38 ±0.06 fold) in the A/J mouse lung than in the

BALB/c mouse lung. The human homolog p63MAPK gene encodes an ERK3-related

kinase (194) and was previously mapped to the human chromosome 18q12-q21(195). In

contrast to the human p44MAPK (ERK1) and p41MAPK (ERK2) genes, much less has

been known about the function of the p63MAPK. The difference in subdomain VIII,

where p63MAPK contains a SEG/SPR motif instead of the TEY/APE found in the ERK

subfamily, the TPY/APE found in the JNK/SAPK subfamily or the TGY/APE found in

the p38/RK subfamily, may suggest a different role for p63MAPK in signal transduction.

Besides the Mapk4 gene, one unknown gene, mCG58197, also exhibited significantly

less expression level in the A/J mouse lung compared to that in the BALB/c mouse lung

(0.36±0.12 fold). This gene encodes a putative GABA-A receptor subunit epsilon-like

protein. Its function remains to be determined.

The human RAD30B was mapped to the human chromosome 18q21.1 and was

suggested as a potential tumor suppressor gene due to its specific chromosome location

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(196). It belongs to the Rad30 branch of the recently described UmuC/DinB/Rev1/Rad30

family of DNA polymerases (196, 197) and encodes a 715-amino-acid DNA polymerase

(DNA POLΙ ) in human . Based upon in vitro studies, DNA POLΙ has the lowest fidelity of any eukaryotic polymerase studied to date, which suggests that its activities must be highly specialized. The fact that POLI orthologues are evolutionarily conserved in higher eukaryotes from Drosophila to humans also suggests that it presumably provides some selective advantage. Several studies show that the RAD30B gene may play a role in the somatic hypermutaion (198, 199), a course in which specific mutations occur as part of antibody diversity. Intriguingly, recent studies suggested that POLI had an intrinsic 5’- deoxyribose phosphate (dRP) lysase activity and might actually function to decrease the mutagenic potential of lesions formed via the deamination of cytosine (200, 201). Our study has revealved that there are 25 nucleotide polymorphisms in Rad30b coding region, causing ten amino-acid alterations. Further function tests will determine if these amino- acid alterations has caused any function difference between A/J and BALB/c Polι proteins.

Mouse SNPs database is a valuable resource to facilitate the discovery of gene mutations and polymorphic markers. The present Celera mouse RefSNP database contains 2,566,706 SNPs based on genomic sequences of five inbred strains of mice.

Investigation on the SNP distribution in the Par2 region gave us a global view of potential functional nucleotide polymorphisms in inbred strains of mice. More

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importantly, further analyses on these SNPs will lead us to narrow the Par2 region and

find new Par2 candidates.

In summary, we have fine mapped the Par2 locus into a region defined by the

marker D18Mit103 and the marker D18Mit162. By real time RT-PCR analyses and

sequence analyses, five genes are now presented as potential Par2 candidate genes, including Rad30b, Myo5b, Smad7, Mapk4 and mCG58197. In the future, SNPs analyses in the Par2 region may further narrow down the Par2 region and reveal other candidate genes. Eventually, in vitro and in vivo studies on these candidate genes will identify the

Par2 gene.

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9 8

Tumor multiplicity 7 (tumors/mouse)

6 P<0.0001 5 4 3

2 1 0 Control strain CC Congenic strain AC

Figure 5.1 Tumor multiplicity of congenic strain AC and control strain CC. The congenic strain AC is heterozygous for the entire 28-cM Par2 region and had a tumor multiplicity of 6.1±1.5 tumors/mouse. The control strain CC is homozygous for the entire

28-cM Par2 region and had a tumor multiplicity of 2.6 ±1.4 tumors/mouse. P value is

<0.0001.

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Figure 5.2 Congenic mapping of the Par2 QTL. The following markers were used to determine Par2 region genotypes: D18Mit34, D18Mit119, D18Mit202, D18Mit36,

D18Mit122, D18Mit207, D18Mit103, D18Mit162, D18Mit155, and D18Mit4.Only the core recombinant regions are shown. The solid box represents a heterozygous genotype

(“H”) at the locus and the open box represents a BALB/c homozygous genotype (“C”) at the locus. For urethane-induced lung tumor bioassays, 5-week-old N9 mice were given a single intraperitoneal (i.p) injection of urethane (1 mg/g body weight) in 0.2 mL phosphate buffered saline (PBS) .The physical position of each marker was derived from the Celera mouse genome (CDS3.6). The Par2 QTL is located between the marker

D18Mit103 and the marker D18Mit162 as determined by the subcongenic strains 275 and

317.

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Tumor multiplicity (per mouse) P value D18Mit207 Par2 QTL D18Mit103 D18Mit162 D18Mit155 Congenic strain AC 6.1±1.5 P<0.0001

Subcongenic strain 423 6.6±1.8 P<0.0001

Subcongenic strain 275 6.0±2.4 P=0.0005 118 Subcongenic strain 271 1.3±1.2 P=0.1519 Subcongenic strain 71 7.7±3.2 P<0.0001

Subcongenic strain 317 5.5±1.7 P=0.0033 P=0.8305 Subcongenic strain 412 2.5±1.2 Control strain CC 2.6±1.4

66.9 68.5 74.8 80.5 Mb

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Figure 5.3 Mouse/Human comparative map for the Par2 candidate region. The physical position for each gene was derived from Celera (CDS 3.6). There are totally 79 known or unknown genes are located in the mouse Par2 candidate region and 36 genes are located in the human 18q21 syntenic region. Homology between a mouse gene and its human counterpart is indicated by line.

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Mouse Par2 Human syntenic candidate region region on 18q21 (Mb) (Mb) hCG1658980 50.7 RAD30B 50.5 68.5 D18Mit103 MBD2 50.4 68.8 Rad30b 68.9 Mbd2

69.5 Dcc DC 48.6

LOC51320 47.4 MADH4/SMAD4 47.3 ELAC1 47.2 mCG51389 ME2 47.1 Madh4/Smad4 B29 47.0 RikcDNA8430417G19 p63MAPK 46.8 71.8 mCG119144 hCG23691 46.6 71.9 CGBP 46.5 72.0 mCG19687 MBD1 46.4 72.1 MYO5 46.1 72.2 Mapk4 ACAA2 46.0 72.4 LIPG 45.8 72.5 RikcDNA2810433K01 mCG22647 RPL17 45.7 Mbd1 FLJ20071 45.3 73.0 Myo5b 45.1 73.2 mCG22649 44.8 73.6 Lipg 73.7 mCG123291 Madh7/Smad7 MADH7/SMAD7 44.2 mCG119038 KIAA0427 74.3 mCG19147 hCG1790951 44.0 74.5 Madh2/Smad2 MADH2/SMAD2 74.8 D18Mit162 HSPC039 43.4 DKFZP564D137 43.3

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Expression Position difference Gene ID Description Primer sequences (Mb) (A/J : BALB/c) Methyl-CpG binding domain Forward: 5'-GCT GTG GCC AGT GCT TTA CA -3' mCG9254 68.85-68.92 0.84±0.03 protein 2 (Mbd2) Reverse: 5'-CCA CGG CAG CAG AGA CTT G-3' GABA-A receptor subunit epsilon- Forward: 5’-GCC AGA TCT CAA AGC CGT TT-3’ mCG58197 71.77-71.81 0.36±0.12 like Reverse: 5’-GGA GAA ATG ACG ATG ACA GGA AA- Drosophila MAD homolog 4 Forward: 5’-GCC GTC CTT ACC CAC TGA AG-3’ mCG19685 71.88-71.93 1.2 (Smad4) Reverse: 5’-CGT TGA TGC GCG ATT ACT TG –3’ Mitogen-activated protein kinase 4 Forward: 5’- AAC GGG CTG GTG CTG TCA -3’ mCG22648 72.15-72.20 0.38±0.06 (Mapk4) Reverse: 5’- GAT CTT CTT CAC GGC CAC CTT -3’ Forward: 5’-GCA CCC GCC TTC AGG AA-3’ mCG22647 72.44-72.46 Rb-binding protein-like 1.2±0.05 Reverse: 5’-CTG CTT AGC GCG AAG AAT GA-3’ Methyl-CpG binding domain Forward: 5’-CCC GCC CTG GTC TCA AG-3’ 121 mCG22652 72.49-72.51 1.35±0.5 protein 1(Mbd1) Reverse: 5’-AAT CAC GTT TAC CCC AAA AAC AG- Forward: 5’-AGC GAA GGA AAG GCG AGA A-3’ mCG123290 72.51-72.59 unknown 0.61 Reverse: 5’-TCC AGA CCC AGG CGT GTA TT-3’

Forward: 5’-AGC TCG AGG TCC GCA AAG A-3’ mCG123286 72.95-73.00 Myosin Vb (Myo5b) 2.15±0.15 Reverse: 5’-GCT GGT CGG CAT TCA TGA T-3’ Forward: 5’-CCA GGT TGG CTA GTC TGT CTG A-3’ mCG48910 73.40 Unknown 0.85 Reverse: 5’-GGC CAC ATT GGC CCT GTA G-3’ Drosophila Mad homolog7 Forward: 5’-CTT ACT CCA GAT ACC CAA TG-3’ mCG9366 73.60-73.63 1.76±0.5 Reverse: 5’-CAG GCT CCA GAA GAA GTT GG-3’ (Smad7)

Table 5.1 Real-time RT-PCR gene expression analyses in the Par2 region revealed some genes differentially

expressed in A/J and BALB/c mouse lung tissue. 121

Amino- acid Polymorphisms Codon A/J BALB/c 4 TTG (Leu) TCG (Ser) 7 GGG (Gly) CGG (Arg) 334 CAG (Gln) AAG (Lys) 378 TCC (Ser) GCC (Ala) 518 TAT (Tyr) TCT (Ser) 524 AAT (Asn) TAT (Tyr) 569 GCT (Ala) GAG (Glu) 574 GAG (Glu) GAT (Asp) 606 CAA (Gln) CGA (Arg) 645 ACA (Thr) ATA (Ile)

Table 5.2. Amino-acid polymorphisms between A/J and BALB/c Rad30b genes

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Number Putative Single Nucleotide Polymorphisms( SNPs) Gene ID of 5’- Mis- Non- 3’-UTR Silent SNPs UTR sense sense mCG9258 3 + + + mCG54865 1 + mCG1033415 8 + + + + + + + + Dcc 7 + + + + + + + mCG1033577 1 + mCG49969 1 + mCG1033576 1 + mCG1033575 1 + mCG1033573 2 + + mCG1033572 5 + + + + + mCG1033342 4 + + + + mCG1033569 1 + mCG58197 4 + + + + mCG1033568 1 + mCG51389 2 + + Smad4 2 + + mCG1033412 3 + + + Rik 8430417G19 11 + + + + + + + + + + + mCG119144 3 + + + mCG1033567 1 + mCG19687 2 + + mCG1033411 2 + + mCG1033562 1 + Rik 2810433K01 4 + + + + mCG22647 2 + + mCG1033561 3 + + + Mbd1 1 + mCG22653 1 + mCG123290 1 + mCG1033560 3 + + + mCG55210 1 + mCG1033409 2 + + Myo5b 2 + + mCG22649 2 + +

Table 5.3 Celera computer-predicted Single Nucleotide Polymorphisms (SNPs) in the Par2 region (continued). Genomic sequences of A/J, 129X1/SVJ, 129S1/SvImJ,

DBA/2J and C57BL/6J inbred strains of mice were used to produce these SNPs.

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Table 5.3: continued.

Lipg 4 + + + + mCG1033558 1 + mCG1033557 4 + + + + mCG123291 1 + mCG9364 5 + + + + + mCG48910 1 + mCG9361 2 + + mCG1033556 1 + Smad7 4 + + + + mCG1033553 2 + + mCG119038 3 + + + mCG1033552 3 + + + mCG1033551 3 + + + mCG57421 7 + + + + + + + mCG19147 3 + + + mCG1033549 3 + + + mCG50780 3 + + + Smad2 3 + + + mCG1033547 7 + + + + + + + mCG1033299 1 +

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CHAPTER 6

FUTURE DIRECTIONS

6.1 Characterization of Pas1 candidate genes

In the present study, we have identified three Pas1 candidate genes Lrmp, Pas1c1 and Pas1c2 in the newly fine- mapped candidate region. Which one of these three genes may be the “real” Pas1 gene will be an immediate question to be answered in our next step. Functional analyses should be performed to characterize these three genes on both levels of In vitro and In vivo. The primary purpose is to classify the Pas1 gene, i.e. an oncogene or a tumor suppressor. Festing et al. analyzed mean tumor numbers in F2 and backcross mice with the Pas1 alleletypes. The result indicated that the genes at the Pas1 locus are acting additively, with heterozygotes being intermediate in tumor number between the two homozygotes. Additional studies on (BALB/c x C3H) F2 population also indicated that the biological activity of the Pas1 locus is under a gene dosage control. Conventionally, it is well recognized that oncogenic effects can be readily affected by gene dosage while loss of function in both alleles is necessary to affect a tumor suppressor. However, recent research on some tumor suppressors (e.g. PTEN gene)

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has demonstrated gene dosage alteration (haploinsufficiency) can also affect function of a tumor suppressor (75-77). Thus, it is still uncertain if the Pas1 gene(s) is an oncogene or a tumor suppressor. Another possibility is that the Pas1/s allele is functionally oncogenic and the Pas/r allele is functionally tumor suppressing. The overall gene effect at the Pas1 locus is determined by interactions between the two alleles.

In vitro anchorage-dependent and anchorage-independent cell growth assays can be sequentially performed following their standard procedures. Anchorage-dependent focus-forming assay depends on the loss of contact inhibition following cell transformation and will be conducted at the first step. Anchorage –independent soft agar cell growth assay is more stringent than focus-forming assay and will be performed after positive results have been shown in the latter. In vivo nude mice tumorigenicity assay will be conducted to further demonstrate gene properties shown in the above in vitro assays.

6.2 Generation of Pas1 mouse models

The Pas1 QTL plays a major role in predisposition of lung tumor susceptibility among inbred mouse strains. After characterization of its oncogenic or tumor-suppressing properties, suitable mouse models can be generated to further demonstrate relationship between the putative Pas1 gene and mouse lung tumor susceptibility. Based on the gene property, various mouse models such as transgenic, knock-out or knock-in can be used.

Recently conditional transgenic mice have been used in elucidation of functions of

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transgenes. By combining a tissue-specific promoter and a doxycycline-dependent tet-on system, one can place expression of a transgene under spatial and temporal control, thus better mimicking real-world gene effects. In mice, the SP-C gene is expressed in type II epithelial cells lining the lung alveoli. It was suggested that mouse lung adenomas arise from alveolar type II cells, although papillary adenomas may arise from bronchiolar

Clara cells. The promoter region of the human SP-C gene can been used to direct the putative Pas1 gene expression (202). Only the gene showing functionally relationship with lung tumor susceptibility can be considered as the real Pas1 gene.

6.3 Functional testing of Rad30b gene and fine mappig the Par2 QTL

Our study has fine mapped the Par2 into a 6.3-Mb candidate region. Although

real time RT-PCR expression analyses and sequence analyses revealed five genes as

potential Par2 candidate genes, their candidacy should be clarified by further functional

analyses. The Rad30b gene carries 25 nucleotide polymorphisms (causing ten amino-acid

changes, see Table 5.2) in its coding region between A/J and BALB/c mice and will be an

immediate subject for functional testing. In vitro studies should be focused on the enzymatic activity of A/J and BALB/c Polι proteins. Further in vivo animal models can

be established after in vitro studies have produced positive results. On the other hand,

fine mapping of the Par2 region will still be needed to reduce the number of potential candidate genes. A major obstacle in fine-mapping Par2 candidate region is lacking

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polymorphic markers between the A/J and BALB/c strains due to their close origin.

Computational SNP analyses have shown that there are 54 various kinds of single nucleotide polymorphisms in putative genes. Testing these SNPs using A/J and BALB/c mice DNA may help us find more polymorphic markers between these two strains.

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