A NEW CD44v3-CONTAINING ISOFORM IS INVOLVED IN TUMOR CELL GROWTH AND MIGRATION DURING HUMAN BREAST CARCINOMA PROGRESSION

Eric D. Kalish, M.D.³, Naoko Iida, Ph.D.¹, Frederick L. Moffat, M.D.², Lilly Y.W. Bourguignon¹

1Department of Cell Biology and Anatomy, University of Miami, School of Medicine, Miami, Florida, ²Division of Surgical Oncology, Department of Surgery, University of Miami School of Medicine, Miami, Florida, ³Department of Surgery, Medical Center of Delaware, Wilmington, Delaware

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Materials and Methods

3.1. Human Breast Carcinoma Samples
3.2. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and One-Step Cloning
3.3. Southern Blot Analysis
3.4. Immunohistochemistry and RT in situ PCR
3.5. Cell Lines
3.6. Stable Transfection
3.7. Analysis of CD44 Expression At The Protein Level
3.8. In vitro Cell Migration Assays
3.9. In vitro Cell Growth Assays

4. Results

4.1. Identification of CD44 Variant Transcripts Expressed in Human Breast Carcinoma Tissues By RT-PCR, Southern Blot Analyses, cDNA Cloning and Nucleotide Sequencing
4.2. Immunofluorescence and RT in situ PCR
4.3. Transfection and Expression of CD44v2,Dv3-10 In Human Breast Tumor Cells (MCF-7) Containing CD44
4.4. Analyses of Tumor Cell Growth and Migration of MCF-7 Transfectants Expressing CD44v2,Dv3-10

5. Discussion

6. Acknowledgment

7. References

1. ABSTRACT

CD44 isoforms belong to a family of cell adhesion molecules expressed on the cell surface of many tumor cells during human breast cancer progression. In this study we have analyzed the expression of CD44v3-containing isoforms [containing heparan sulfate addition sites for growth factor binding] in primary breast tumors, axillary nodal metastases and normal breast tissue. Using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot, cloning, nucleotide sequencing and RT-in situ-PCR analyses, we have found that at least two CD44v3-containing isoforms, including one new species of CD44v2,Dv3-10 (Dv3 defined as a v3 exon lacking the first 24 base pairs) and another previously reported CD44v3,8-10 are preferentially expressed in human primary breast tumor and axillary nodal metastases but not in normal breast tissues. These finding suggest that these CD44v3-containing isoforms are closely associated with breast cancer metastasis.