Kazuyuki Matsushita¹, Takeshi Tomonaga¹, Toshiko Kajiwara¹, Hideaki Shimada⁵, Sakae Itoga¹, Takaki Hiwasa³, Shuji Kubo⁴, Takenori Ochiai², Hisahiro Matsubara², Fumio Nomura¹

1Departments of Molecular Diagnosis & Division of Clinical Genetics and Proteomics, ²Department of Frontier Surgery, and ³Biochemistry and Genetics, Chiba University, Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 , ⁴Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya-shi, Hyog, ⁵Department of Gastrointestinal Surgery, Chiba Cancer Center, 666-2 Nitona, Chuo-ku Chiba-shi, Chiba

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Materials and methods

3.1. Plasmids

3.2. Human tissue samples

3.3. Immunocytochemistry

3.4. Apoptosis detection

3.5. Protein extraction and immunoblotting

3.6. Reverse transcriptase (RT)-PCR and real-time quantitative PCR

3.7. MTT assay

3.8. FIR (FBP-interacting repressor) adenovirus vector

3.9. Tumor xenografts in animal model experiments

4. Results

4.1. The amino terminal domain of FIR is required to represses endogenous c-Myc expression

4.2. FIR-induced apoptosis is prevented by enforced expression of c-Myc

4.3. FIR is paradoxically upregulated in colorectal cancer correlating with increased c-Myc

4.4. An alternatively spliced form of FIR is expressed in tumors, but not the adjacent normal tissue

4.5. FIR adenovirus indicates antitumor effect on human cancer cells in vitro and in vivo

5. Discussion

6. Acknowledgements

7. References

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.