We further investigated the structural and morpho-
logical changes in senescent PLXSNp53 NIH3T3 cells
(shown in Plateĉ-2). After the senescence was triggered
in PLXSNp53 NIH3T3 cells, nuclear and cellular size
increased; then cytoplasm spread and formed some pro-
tuberances; subsequently cells multinucleated; cytoplasm
vacuolated; nuclei approached cytoplasmic membrane and
were excluded; eventually cells were lysed. In this process,
lipofuscin vesicles increased gradually in cytoplasm.
These morphological alterations accorded with those in
the replicative senescent cells[14], which demonstrated
further that overexpression of p53 activated the senes-
cence program in NIH3T3 cells.
Fig. 4. Expression of p53 was detected in NIH3T3 cells, PLXSN
NIH3T3 cells and PLXSNp53Val135 NIH3T3 cells by Western blotting.
1, NIH3T3 cells; 2, PLXSN NIH3T3 cells; 3, PLXSNp53Val135
NIH3T3 cells.
It is possible that p21 up-regulated by overexpres-
ü
18]
sion of p53[5,16
functions in the NIH3T3 cells senes-
cence, which requires more investigations. Since NIH3T3
cells are immortal mouse embryonic fibroblasts, which
are different from tumor cells and from primary cells, can
the overexpression of p53 induce a rapid senescence of
these cells? It is necessary for cells to break through the
restriction of cellular senescence mechanism to proliferate
during their carcinogenesis[6]. So the mechanism of se-
nescence has been destroyed in tumor cells. Only these
tumor cells in which the downstream pathway of p53
maintained integral in senescence mechanism can be in-
duced rapidly into senescence by overexpression of p53.
On the contrary, since there is an integral senescence
mechanism in primary cells[6,14], it is suggested that over-
expression of p53 can induce a rapid onset of senes-
cence, which also requires further investigations.
Fig. 5.
Growth curves of NIH3T3, PLXSN NIH3T3 and
PLXSNp53Val135 NIH3T3 cells in DMEM-10 at 32ć. 1, NIH3T3
cells; 2, PLXSN NIH3T3 cells; 3, PLXSNp53Val135 NIH3T3 cells.
(π) Overexpression of p53Val135 in NIH3T3 cells
triggers a rapid senescence. Normal cells deviate from
cell cycle, irreversibly lose the ability of proliferation and
enter a comparatively stable state, which is termed repli-
cative senescence. Compared with cells in other state,
senescent cells still remain active metabolically, but there
are many fundamental changes in morphology or function,
such as the insensitivity to mitogen stimulation and resis-
tance to apoptosis [6,13,14]. In senescent cells, the activity of
E-galactosidase increases obviously, which can be de-
tected by SA-ꢁE -Gal staining. But SA-ꢁE -Gal activity is
undetectable in young cells which have the ability to pro-
liferate or which is in the nondividing state by withdrawal
of growth factors. So SA-ꢁE -Gal is a good marker of se-
nescence in some cells[15]. Shown in Plateĉ-1, PLXSN
p53 NIH3T3 cells were stained blue (positive) by SA-ꢁE
-Gal after incubating at 32ć for 10ü14 d, showing that
PLXSNp53 NIH3T3 cells entered senescent phase. How-
ever, under the same condition, negative results of SA-ꢁE
-Gal staining were obtained in NIH3T3 cells and PLXSN
NIH3T3 cells, showing that these kinds of cells did not
enter senescent phase. In summary, we made a conclusion:
when PLXSNp53 NIH3T3 cells were transferred from
37.5ć to 32ć, plenty of p53Val135 mutant converted
into wild type conformation, activated the senescent pro-
gram, and finally resulted in cellular senescence.
Acknowledgements We thank Prof. Moshe Oren in Weizman Institute
of Israel for providing the plasmids PLXSNp53 NIH3T3 and PLXSN,
and Dr. Wu Xiaobing with the Chinese Academy of Medical Sciences for
providing packaging cell line GP+E-86. Also we thank Dr. Tao Wei,
Zhao Yun, Liu Xiaoling and others in our lab for their help. This work
was supported by the National Natural Science Foundation of China
(Grant No. 39300075) and the National Basic Sciences Research Pro-
gram (Grant No. G19990539).
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