Gene expression patterns associated with the metastatic phenotype in rodent and human tumors.

Using subtractive technology, we have generated metastasis-associated gene expression profiles for rat mammary and pancreatic adenocarcinomas. Several genes whose expression is thought to be related to tumor progression such as c-Met, urokinase-type plasminogen activator receptor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones showed no significant homology to genes with known function. Notably, several of the metastasis-associated clones were also expressed in metastatic lines but not in nonmetastatic lines of other tumor models. Furthermore, in situ hybridization using selected clones documents the relevance of these results for human cancer because strong expression in tumor cells including metastases was detected in human colorectal cancer samples and, to a lesser extent, in mammary cancer samples. These data support the concept that tumors express a "metastatic program" of genes.

Functional genomics in cancer research: identification of target genes of the Epstein-Barr virus nuclear antigen 2 by subtractive cDNA cloning and high-throughput differential screening using high-density agarose gels.

In the past, the identification and isolation of phenotype-associated genes was a difficult and time-consuming task. However, recent improvements of methods that are designed to isolate differentially expressed genes have remarkably speeded up the process of target gene isolation. The ultimate goal of functional genomics is to apply these technologies to clone phenotype-associated genes irrespective of the availability of probes (e.g., antibodies) and an intimate knowledge of biological background. We demonstrate the use of a novel subtractive cDNA cloning approach for the isolation and characterization of target genes of the Epstein-Barr virus nuclear antigen 2 (EBNA2). Two different subtractive cDNA libraries specific for two different time periods following activation of a conditional estrogen receptor/EBNA2 (ER/EBNA2) fusion protein were generated. Comparison of the two libraries by cross-hybridization experiments allowed the differentiation between direct and indirect target genes of EBNA2 and led to the identification of a novel direct target gene of EBNA2.

Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.

A high throughput screening for rarely transcribed differentially expressed genes.

A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined.