Περίληψη:
Background: 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
protooncogene. 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 dimethylbenzanthracene (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.
Materials and Methods: 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.
Results: 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.
Conclusions: 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.
Συγγραφείς:
Katsanakis, KD
Gorgoulis, V
Papavassiliou, AG
Zoumpourlis,
VK
