Fig. 2.

ARF, but not p53, is activated in REF52 cells transformed by PyMT and PyST. (A) REF52 cells transformed by wild-type Py DNA (PyREF52) express PyLT, PyMT, and PyST, whereas cells transformed by the bc1051 mutant DNA (bc1051 REF52), which contains a mutation in the LT splice donor site (22), only express PyMT and PyST. Shown is Western analysis of extracts of two different isolates of transformed PyREF52 and three different isolates of bc1051 REF52 cells probed with the PYC antibody that detects common amino acid sequence in PyLT (LT), PyMT (MT), and PyST (ST). (B) REF52 cells transformed by only PyMT and PyST (bc1051 REF52) contain ARF. Western analysis of untransformed REF52 cells, two different isolates of PyREF52 cells transformed with wild-type Py expressing PyLT, PyMT, and PyST, and two different isolates of cells transformed with the bc1051 mutant that only expresses PyMT and PyST (bc1051 REF52) (see A) were probed with the rabbit anti-Rat ARF polyclonal serum 821B6. The differences in the amounts of ARF are most likely due to differences in the levels of the PyMT-induced cellular signaling that activates ARF in the different Py-transformed cell clonal isolates. (C) REF52 cells transformed by either Py wild-type (PyREF52) or the bc1051 mutant (bc1051 REF52) DNA express low levels of wild-type p53 that can be activated by DNA damage. Cells from one line of transformed PyREF52 cells, one isolate of transformed bc1051 REF52 cells, and untransformed REF52 cells were cultured in the presence or absence of the DNA-damaging agent Adriamycin for 7 h before being assessed by Western analysis for the presence of p53, p21, and MDM2.