Transfection of metastatic capability with total genomic DNA from human and mouse metastatic tumour cell lines

Hayle, Allan J.; Darling, Dawn L.; Taylor, Anne R.; Tarin, David

doi:10.1111/j.1432-0436.1993.tb01600.x

Cited by 11 publications

(8 citation statements)

References 41 publications

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“…In earlier studies, human DNA transfected into 3T3 cells has been recovered by identification of human specific Alu sequences (Gate et al, 1995;Hayle et al, 1993). Although several oncogenes have been identified by this method, the risk involved in this strategy is that, although Alu sequences are scattered throughout the human genome they are not contained in every fragment of human DNA (only 50% according to probability and depending on the sizes of the fragments).…”

Section: Discussionmentioning

confidence: 99%

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

Beesley²,

Smith³

et al. 1998

Br J Cancer

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Summary Prostate cancer is the second leading cause of male death from malignant disease in Europe and in the USA. Failure to prevent or eliminate metastatic dissemination is a fundamental problem underlying the current inadequate treatment of prostate cancer, and novel therapeutic strategies are required if this disease is to be successfully managed. No independent markers are yet available to predict the behaviour of any individual prostate cancer, particularly its potential to metastasize, and there is now an urgent prerequisite to identify and characterize genes specifically involved in determining the metastatic phenotype of prostate cancer cells before any biologically appropriate treatment modality can be devised. To identify DNA sequences that trophically promote the metastatic phenotype, we have established a new transfection assay with which to monitor activity of prostate cancer genomic DNA. Rat prostatic G and AT6.1 cell lines derived from the same original Dunning R3327 rat prostatic carcinoma exhibit, respectively, low-and high-metastatic phenotypes when grown in syngeneic Copenhagen rats. Rat mammary epithelial cell line 'Rama 37' derived originally from Wistar-Furth rats yields benign non-metastasizing adenomas when inoculated subcutaneously into syngeneic animals. In this report, the Rama 37 cell line is successfully used as the recipient cell-line for transfected DNA fragments extracted from rat prostatic carcinoma G and AT6.1 cells. New metastatic variants of Rama 37 cells have been generated. Enzymatically fragmented genomic DNA from rat metastatic prostate carcinoma cell lines was co-transfected together with plasmid pSV2neo into parental Rama 37 cells, followed by culture in the presence of Geneticin-G418 to select for the transfected cells. To enable subsequent identification of metastasis-promoting DNA sequences, the fragmented genomic DNA sequences were covalently attached to specifically engineered linker DNA molecules to flank the genomic DNA before transfection. Thereafter, the resulting transfectants were pooled and inoculated into mammary fat pads of female Wistar-Furth rats. Metastases produced by the transfectant cells in vivo were reestablished from secondary tumours and probed for the presence of the specific synthetic oligonucleotide sequences that flanked, and hence identified, the presence of the transfected DNA. These new metastatic cells are shown to provide a sensitive assay system with which to detect DNA sequences responsible for conveying the metastatic phenotype of prostate cancer when inoculated into syngeneic rats.Keywords: DNA transfection; Dunning prostatic carcinoma cells; Rama 37 cell-line; metastatic phenotype Adenocarcinoma of the prostate is now the most common human non-cutaneous malignant neoplasm to affect men. In the USA and Europe, it is the second leading cause of male deaths from malignant disease after lung cancer (Foster, 1990;Boring et al, 1994). Despite the increasing incidence of this disease, current knowledge of molecular mechanisms underlying pr...

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Section: Discussionmentioning

confidence: 99%

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

Beesley²,

Smith³

et al. 1998

Br J Cancer

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“…There have been a number of reports in which a spontaneous metastatic phenotype was successfully transferred into normal mouse or rat fibroblasts, or non-metastatic mouse tumor cell lines by transfections involving metastatic human tumor cell DNA and an appropriate selectable marker gene [1][2][3][4][5][6][7][8]. However, in spite of apparent successful acquisition of metastatic competence by the recipient cells, such experiments have not yet led to the identification of novel metastasis-associated genes.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, because of the transient nature of the transfected neo-resistance gene, the phenotypic behavior exhibited by the re-established metastatic cell lines could not be attributed conclusively to co-transfected donor DNA sequences. In the final study [1], although experimental metastases (lung colonization following intravenous tumor cell injections) at levels very similar to those caused by the primary transfectants were repeatedly seen following similar injections of successive generations of metastatic cells re-established in culture, the same cells when injected subcutaneously did not produce spontaneous metastatic foci even though tumors developed at the injection sites. This implied that only a portion of the criteria for metastatic progression was being met.…”

Section: Introductionmentioning

confidence: 99%

Acquisition and enhanced expression of the metastatic phenotype following transfections of genomic mouse tumor DNA containing human SCLC gene sequences

1995

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Previous primary and secondary co-transfections of genomic DNA from a metastatic human small cell lung cancer cell line into NIH/3T3 cells resulted in a murine fibrosarcoma cell line (Tx93B) that produced frequent spontaneous lung metastases in subcutaneously injected tumor-bearing nude mice. In order to transfer the acquired metastatic behavior to additional cell lines that could then be tested in syngeneic immunocompetent animals, DNA from Tx93B cells was transfected without additional neo gene into Balb/c embryo fibroblasts, which led to the isolation of a tertiary transfectant cell line (D3) of low spontaneous metastatic potential in normal Balb/c mice. Subsequent cell lines established serially from lung metastases in mice injected with D3, and metastatic descendants of D3 (all selected for the original neo marker in G-418), resulted in three generations of metastatically variant cell lines capable of causing pulmonary metastases in 11.1%, 54.6%, and 89.5%, respectively, of subcutaneously injected animals, and in 100% of normal mice injected intraperitoneally. There was no apparent ras-family oncogene participation in the metastatic behavior of either of the two DNA donor cell lines or in the metastatically variant tertiary transfectants. Gelatin zymography indicated that the secretion of gelatinolytic enzymes in vitro by the variant cell lines was inversely proportional to their metastatic capability. Human Alu repeat gene sequences detected in the metastatic variants suggested that co-transfected metastasis-associated genes present in the original human DNA donor cell may have contributed to acquisition of the metastatic phenotype by the tertiary transfectant cell lines. The increase in metastatic potential observed in successive generations of the D3-derived tumor cell lines, further suggested that selection for cells having increased metastatic capability had occurred during passage in vivo accounting for the phenotypic change. Because of their common origin and progressively metastatic nature these cell lines may prove useful in the identification of metastasis-associated genes accessible through the use of differential expression cloning strategies.

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“…Such findings indicate that pulmonary metastasis after inoculation, either s.c. or mfp, or with Matrigel, primarily depends on intrinsic properties of the tumour cells (Fidler, 1978;Tarin & Prince, 1979), but can be modulated by local microenvironmental factors. Recent results (Steeg et al, 1988;Hayle et al, 1993) indicate that metastatic events occur as a result of genetic disturbances which allow the inappropriate expression of genes that are silent in most cells, enabling the cells affected and their progeny to disseminate from the primary site. This new evidence suggests that metastasis may occur as a consequence either of failure of a negative regulatory event responsible for inhibiting inappropriate cell migration and distant colonisation, perhaps involving the nm23 gene (Steeg et al, 1988), or of the activation and up-regulation of a gene capable of dominantly conferring the phenotype (Hayle et al, 1993).…”

Section: Matrigelmentioning

confidence: 99%

“…Recent results (Steeg et al, 1988;Hayle et al, 1993) indicate that metastatic events occur as a result of genetic disturbances which allow the inappropriate expression of genes that are silent in most cells, enabling the cells affected and their progeny to disseminate from the primary site. This new evidence suggests that metastasis may occur as a consequence either of failure of a negative regulatory event responsible for inhibiting inappropriate cell migration and distant colonisation, perhaps involving the nm23 gene (Steeg et al, 1988), or of the activation and up-regulation of a gene capable of dominantly conferring the phenotype (Hayle et al, 1993). In any event, once this balance has been disturbed, it appears that microenvironmental influences, such as the site of growth of the tumour cells or the constitution of the adjacent tissue matrix, can accelerate tumour growth and dissemination.…”

Section: Matrigelmentioning

confidence: 99%

Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells

Bao

Matsumura²,

Baban³

et al. 1994

Br J Cancer

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Transfection of metastatic capability with total genomic DNA from human and mouse metastatic tumour cell lines

Cited by 11 publications

References 41 publications

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

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

Acquisition and enhanced expression of the metastatic phenotype following transfections of genomic mouse tumor DNA containing human SCLC gene sequences

Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells

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