Generation Of Metastatic Variants Research Articles

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

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.

Generation of Metastatic Variants by Transfection of a Nonmetastatic Rat Mammary Epithelial Cell Line with DNA from a Metastatic Rat Mammary Cell Line

Transfection of rat mammary (Rama) 37 epithelial cells, which yield nonmetastasizing adenomas in syngeneic Wistar-Furth rats, with HindIII-fragmented cellular DNA and the drug-resistance plasmids pSV2gpt or pSV2neo yields drug-resistant transformants with a frequency of 10(-4)-10(-5). Transformant cell lines transfected with the following, pSV2gpt alone, pSV2gpt and Rama 37 DNA, pSV2gpt and DNA from a metastasizing cell line Rama 800 (CT set), pSV2neo and salmon sperm DNA, pSV2neo and Rama 800 DNA (C set), all yield tumors when injected subcutaneously into syngeneic rats. A few transformants obtained by cotransfection with DNA from Rama 800 cells produce metastases in lungs and/or lymph nodes. The incidence of such metastases for two transfectants, termed CT4-41 and C18P, is significant at 20 and 24%, respectively, but only half (48%) that achieved with Rama 800 cells. Reintroduction into rats of cells cultured from a metastatic tumor of CT4-41 and of C18P, and from their lung or lymph node metastases, produces either a similar incidence (20-24%) or a significantly higher (48-52%) incidence of metastasis than that of the original transfectants. Cells cultured from nonmetastatic tumors fail to produce any metastatic lesions. When [32P]-labeled gpt or neo DNAs are hybridized to EcoRI-digested cellular DNA of the CT4-41 or C18P series of cell lines, tumors or metastases, gpt binds to one major fragment of 3,800 basepairs, and neo to two major fragments of 5,700 and 4,200 basepairs. The same cell lines produce hybridizing mRNAs of 1,500 and 1,900 bases for the CT4-41 series and 2,000-2,400 bases for the C18P series. It is suggested that transfection of DNA from the metastatic cells causes the nonmetastatic cells to become metastatic, in a genetically dominant manner, but additional steps are required for this process to become established and expressed at a level equivalent to that of the original, metastasizing donor cells.

Differences in organization of metastatic and nonmetastatic tumors initiated by the same B16 melanoma clone in mature and young mice

Subcutaneous transplants of mouse B16 melanoma clone G3.26 grow more slowly, and are markedly more metastatic to the lungs, in mature (greater than 12-month-old) mice than in young (2-month-old) mice. Previous studies suggested that tumors in young mice fail to disseminate viable tumor cells into the hematogenous circulation. To determine if changes in intratumor organization might accompany this altered tumor behavior, G3.26 tumors growing in young and mature mice were examined comparatively at progressive sizes relative to the onset of metastatic dissemination in the older mice. Although the degree of necrosis was comparable in both groups of tumors, vascular density, measured morphometrically in histological sections, was significantly lower in tumors from mature mice at a size when dissemination would be occurring. With the onset of reduced vascular density in tumors in mature mice, there was a substantial increase in the proportion of viable tumor cells that was hypoxic, based on radioresistance and incorporation of the hypoxic cell sensitizer, misonidazole. Quiescent tumor cells, identified by flow cytometry, were also more numerous in tumors from mature mice than in tumors from young mice. Although the importance of these differences in tumor organization to enhanced metastatic behavior is unclear, increased intratumor hypoxia might promote generation of metastatic variants. Alternately, dissemination of tumor cells might be facilitated through a reduced and possibly defective vasculature.

In vivo tumour × host cell fusion in spontaneous syrian hamster metastasis

The HSV-2 tumour system was originally derived from an in vitro transformation of HEF by inactivated HSV-2. When injected s.c. these cells produce spindle-cell sarcomas which are metastatic at a low level. Detailed cytogenetic studies have provided evidence of tumour × normal host cell fusion in two of seven cell lines derived from metastatic lung deposits (Met D and Met G). This is the first report, in an unselected, intraspecific system, of in vivo cell fusion in spontaneous metastatis. The cells of Met D consisted of a heterogeneous population of fused and unfused tumour cells, whereas those of Met G were a homogeneous population of hybrid cells. Fusion, therefore, is likely to have occurred after metastasis in Met D and prior to, or at, metastasis in Met G. The fused cells of Met D showed comparatively little chromosome loss, while in Met G there was loss of approaching one haploid set of chromosomes. The generation of metastatic variants by cell fusion contributes to genetic diversity and emphasizes the importance of tumour heterogeneity in malignancy.

Generation of metastatic variants in populations of mutator and amplificator mutants.

Genetic instability has been hypothesized by P. C. Nowell and other investigators to be an important aspect of tumor progression that leads to the generation of metastatic variants. In this study we examined the rate of generation of metastatic variants in mutant cell lines having increased rates of spontaneous mutation and gene amplification. Parallel clonal populations of the spontaneous mutation rate mutant thy-49 and the gene amplification mutants YMP1 and YMP7 and their respective wild types were generated and grown to a critical population size. The number of metastatic variants in each clonal population was then determined following iv injection into nude mice. Lung tumors were scored 3-4 weeks after injection of cells, and the mean number per clonal population was determined. Analysis of the means with the Luria-Delbruck fluctuation test showed no significant differences in the rate of generation of metastatic variants produced in the genetically unstable lines compared to their normal counterparts. This study suggests that increased spontaneous mutation and gene amplification rates in mammalian cells are not sufficient on their own to increase the rate of generation of metastatic variants.

Dynamic heterogeneity: experimental metastasis studies with RIF-1 fibrosarcoma.

We have examined the metastatic behaviour of cells from a series of clones of RIF-1 fibrosarcoma, a recently derived murine tumour. The clones were grown to different sizes (small: 10(5)-10(6) cells and large approximately 2 x 10(7) cells) and their metastatic potential was quantified using an experimental metastasis assay. There was significant variability between the metastatic potential of clones derived from the same population. Furthermore large clones had higher metastatic efficiency (metastases/cell injected) than the small clones derived from the same population. The results from these experiments indicate that metastatic variants are generated during the growth of the clones. The mean effective rate of generation of metastatic variants was estimated using Luria-Delbruck fluctuation analysis to be 1.7 x 10(-5)/cell/generation. The data described are consistent with a dynamic heterogeneity model of metastasis, in which the variant phenotypes are postulated to arise in growing tumour cell populations at a high rate but need not be stable in order to produce metastases. The results thus indicate that the model is applicable to a recently derived tumour as well as the extensively transplanted KHT sarcoma, B16 melanoma and OTF9 embryonal cell carcinoma.

The generation of metastatic variants by cell fusion

The generation of metastatic variants by cell fusion