Abstract
The ras family of proto-oncogenes encodes for small GTPases that play critical roles in cell-cycle progression and cellular transformation. ERK1/2 MAP kinases are major ras effectors. Tumors in chemically treated mouse skin contain mutations in the Ha-ras proto- oncogene. Amplification and mutation of Ha-ras has been shown to correlate with malignant progression of these tumors. Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. PDVC57 cells were selected from PDV cells, which were transformed with dimethyl-benzanthracene (DMBA) in vitro and then transplanted in adult syngeneic mice. The PDV:PDVC57 pair contains ratio of normal:mutant Ha-ras 2:1 and 1:2, respectively. This genetic alteration correlates with more advanced tumorigenic characteristics of PDVC57 compared to PDV. The squamous carcinoma B9 cell clone was isolated from the same primary tumor as A5 spindle cell line. The mutant Ha-ras allele, also present in B9, is amplified and overexpressed in A5 cells. Therefore these cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis. The ERK1/2 status in the above mouse cell lines was examined by using various molecular techniques. For the study of the tumorigenic properties and the role of the ras/MEK/ERK1/2 pathway in the cell lines mentioned, phenotypic characteristics, colony formation assay, anchorage-independent growth, and gelatin zymography were assessed, after or without treatment with the MEK inhibitor, PD98059. ERK1/2 phosphorylation was found to be increased in PDVC57 when compared to PDV. This also applies to A5 spindle carcinoma cells when compared to squamous carcinoma and papilloma cells. The above finding was reproduced when transfecting human activated Ha-ras allele into PDV, thus demonstrating that Ha-ras enhances ERK1/2 signaling. To further test whether ERK1/2 activation was required for growth we used the MEK-1 inhibitor, PD98059. The latter inhibited cell proliferation and anchorage-independent growth of squamous and spindle cells. In addition, PD98059 treatment partially reverted the spindle morphology of A5 cells. These data suggest, for the first time, that oncogenicity and the degree of progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling.
Highlights
The ras proto-oncogene, which is found mutated in 30% of human tumors and higher in certain tumor types, is involved in tumor development in rodents [1]
It has been previously shown that a reduction in the ratio between normal:mutant Ha-ras is associated with tumorigenicity in squamous cell carcinomas [4]
Injection of PDV cells into nude mice gives rise to tumors only in about 15% of injection sites, whereas PDVC57 is more strongly tumorigenic, with tumors diagnosed as squamous carcinomas arising at all injection sites [4]
Summary
The ras proto-oncogene, which is found mutated in 30% of human tumors and higher in certain tumor types, is involved in tumor development in rodents [1]. During the squamous-spindle stage a genotypic difference occurs in the normal:mutant Ha-ras alleles ratio [4] (Fig. 1). The altered ratio of normal:mutant Ha-ras is maintained in the spindle stage of the carcinogenic process and is strongly associated with a more tumorigenic and invasive cell behavior [5] (Fig. 1). We detected increased expression of the total Ha-Ras content in the clones compared to PDV and PDVC57 cell lines (Fig. 2c). Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. The mutant Ha-ras allele, present in B9, is amplified and overexpressed in A5 cells These cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis
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