Isolation of and effector for metastasis-inducing DNAs from a human metastatic carcinoma cell line

Abstract

Benign rat mammary epithelial cells transfected with restriction enzyme-fragmented DNA from a human malignant metastatic cell line (Ca2-83) produces transfectants that yield metastatic tumours in syngeneic rats. The six metastasis-inducing DNAs (Met-DNAs) that have been isolated from such transfectants are subgene in size and do not code for any expressed mRNAs, but correspond to potential regulatory regions of human DNA from malignant, metastatic cells. In pilot studies the one Met-DNA tested is detectable in some human breast tumours but not in normal tissue. Transfection of all six Met-DNAs singly into the benign mammary epithelial cells causes enhanced expression of osteopontin, whilst transfection of cDNA for osteopontin also induces the metastatic state. These results show that short regulatory DNAs exist in human cancer cells that can induce metastatic spread via a common eector gene, osteopontin, in model rat mammary cell lines.

An optimum anti-melanoma response in mice immunized with fibroblasts transfected with DNA from mouse melanoma cells requires the expression of both syngeneic and allogeneic MHC-determinants

“…[23][24][25][26][27][28][29][30][31] These studies indicated that transfection of genomic DNA from one cell type to another resulted in integration of the exogenous DNA and stable expression of the trans-ferred genes. This was the case for genes specifying missing enzymes, as well as for genes specifying membraneassociated determinants.…”

Generation of metastatic variants by transfection of a rat non-metastatic epithelial cell line with genomic DNA from rat prostatic carcinoma cells

“…Earlier work on the generation of metastatic variants of Rama 37 cells by transfection of genomic DNA from rat and human breast carcinoma cells confirmed the validity of DNA transfection as a technique with which to assay the metastatic capability of genomic British Journal of Cancer (1998) 77(2), 287-296 DNA fragments (Jamieson et al, 1990a;Davies et al, 1994), as well as that of a variety of cellular oncogenes (Bernstein and Weinberg, 1988), and the gene for calcium-binding protein p9Ka (Jamieson et al, 1990b;Davies et al, 1993). Using this technique, several DNA fragments closely associated with metastasis in human breast cancer cell lines have been identified (Chen et al, 1997). In this system, the transfected DNA responsible for inducing metastasis does not code for growth-promoting oncogenes such as ras etc.…”

“…Conversely, transfection of similarly fragmented DNA from benign, non-metastasizing mammary epithelial cells does not produce new cell strains with metastatic ability (Davies et al, 1994). Thus, it is transfection of a particular DNA sequence, and not the transfection process itself, that is responsible for the generation of metastatic variants in this model (Chen et al, 1997).…”

“…The Dunning R3327 rat prostate carcinoma cell model is ideal for examining different aspects of prostate cancer and metastasis as cells exhibiting distinct behavioural phenotypes have been derived from a single common origin, and many characteristics of the system are similar to those of the human disease (Isaacs et al, 1986). Using molecular biological techniques to 'tag' the fragmented donor DNA before transfection (Chen et al, 1997), together with the use of a reliable in vivo assay to detect spontaneous metastases, we have successfully identified fragments of donor prostatic DNA that become integrated into the genome of the resultant transformants. These transformants yield metastatic tumours at frequencies of 8.7% (G cell donor DNA) and 25% (AT6.1 cell donor DNA) respectively.…”

“…The metastatic nature of these new cell lines has been further confirmed by in vivo assay. As the transfected prostatic genomic DNA was marked by flanking sequences of synthetic oligonucleotides (Chen et al, 1997), these metastatic variants of Rama 37 cells now provide a unique resource with which genomic DNA 1 ; *****PCR primer sequence******[ 5' -AAT CCA AGC TTG CGG CCG ATC AGG CCG AAT ATG CGG CCG CAT 3-A GGT TCG AAC GCC GGC TAG TCC GGC TTA TAG GCC GGC GTA ATA TCG A-5 Hindlll ( Induction of metastasis by rat prostatic cancer DNA 289 (Jamieson et al, 1990a;Davies et al, 1994). Exponentially growing parental Rama 37 cells were harvested by scraping and seeded at a density of 0.5-0.75 x 106 cells per 10 ml of Dulbecco's modified Eagle medium (DMEM) in each 10-cmdiameter Petri dish.…”