Frameshift mutations at coding mononucleotide repeats of the hRAD50 gene in gastrointestinal carcinomas with microsatellite instability.

Microsatellite instability (MSI) and frameshift mutations in genes containing nucleotide repeats have been reported in a subset of colorectal and gastric carcinomas. This study describes the analysis of MSI-positive colorectal (39 cases) and gastric carcinomas (36 cases) for the presence of frameshift mutations of the six genes known to be involved in DNA repair and containing mononucleotide repeats in their coding region. Our mutational study of the 75 MSI-positive tumors revealed frequent mutations in hRAD50 (23 cases, 31%), BLM (16 cases, 21%), and hMSH6 (16 cases, 21%); rare mutations in BRCA1 (1 case, 1%) and ATM (3 cases, 4%); and no mutation in NBS1. In contrast, no frameshift mutation was found in 60 MSI-negative colorectal and gastric carcinomas. The mutation of hRAD50, a gene that is involved in the response to cellular DNA damage and forms a complex with hMRE11 and NBS1, has not been reported previously. Our results suggest that frameshift mutations of hRAD50, BLM, and hMSH6 are selected and play a role in the tumorigenesis of colorectal and gastric carcinomas with MSI. The MSI targeting of the hRAD50 and BLM genes represents an additional link between MSI and DNA repair because alteration of these genes could accelerate defective DNA repair.

Crucial to the prognosis of cancer patients is not growth of the primary tumor, but rather dissemination of neoplastic cells to other organs As the process of the activation and inactivation of genes that are involved in tumorigenesis and metastasis is still poorly understood, the aim of this study is to identify novel mammary cancer progression and metastasis genes in vivo. Proviral insertions of mouse mammary tumor virus (MMTV) in mammary epithelial cells are able to activate flanking oncogenes, leading to mammary tumor induction. Classical examples of MMTV-induced oncogenes are Wnt1 and Fgf3. Full neoplastic transformation to an invasive and metastasizing tumor requires activation of collaborating onco/metastasis genes. Thus, additional proviral insertions may lead to metastasis-inducing genes. In this study we used a BALB/c+ mouse strain (ie a BALB/c substrain that aquired C3H-MMTV by forster-nursing), and compared extra proviral integrations in a series of sets of independent primary tumors and metastases. As a first step, the isolated tumor sets (primary tumor and metastases) were analyzed by Southern blotting using a MMTV-LTR specific probe. A number of the lung metastases indeed carried additional MMTV integrations, which were not found in the primary tumor. These additional integrations might activate genes being responsible for the lung metastases. To analyze the flanking sequences more efficiently, an adaptor ligation-mediated PCR (Splinkerette-PCR) was modified for the metastasis-related proviral MMTV integrations. To this end, genomic DNA was digested and ligated to a suitable splinkerette linker. The subsequent PCR gets its specificity by using a unique MMTV-LTR-related oligonucleotide and a splinkerette-specific oligonucleotide, which is only able to bind to the DNA if extension of the MMTV oligonucleotide occurs. BLAST/NIX (DNA analysis software) analysis of the derived additional sequences from 23 tumor sets resulted in the discovery of a novel common integration site. The effect of this putative metastasis gene on the metastic potential of mammary tumor cells is presently being investigated.