Download Comparative Studies of Apoptosis in Xenopus laevis and

ATLA 41, 211–218, 2013
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Comparative Studies of Apoptosis in Xenopus laevis and
Mouse Thymoma Cell Lines
Rachel O. Johnson,1 Thomas V. Tittle,1 Maria P.M. Sefchick,1 Leslie D. Zettergren,1 Laurens N.
Ruben,1 Richard H. Clothier2 and Michael Balls2
1Reed
College, Portland, Oregon, USA; 2c/o FRAME, Nottingham, UK
Summary — With the use of in vitro methods and cell lines, functional aspects of apoptosis in the Xenopus
laevis B3/B7 and mouse EL4 thymoma cell lines are revealed. Moreover, by using information gleaned from
digital imaging and immunocytochemistry, changes in locations of key proteins implicated in apoptotic
anti-cancer responses, e.g. p53 and Mdm2, are shown. Suggestions are offered as to what these results
might mean with respect to the evolutionary conservation of the function and structure of these two molecules and to cancer resistance in amphibians. Finally, studies are described on resveratrol as an anti-cancer therapeutic reagent in the two thymoma cell lines and in normal X. laevis thymocytes.
Key words: amphibians, cancer, digital imaging, FACS analysis, immunostaining, mammalian.
Address for correspondence: Laurens N. Ruben, Department of Biology, Reed College, Portland, OR
97202-8199, USA.
E-mail: [email protected]
Introduction
The evolutionarily conserved protein transcription factor, p53, is found mutated or absent in
half of all human cancers (1). While in mammalian cells, the p53 protein is induced to form as
a consequence of DNA damage, its equivalent in
Xenopus laevis (2) is found to be constitutively
expressed. The constitutive Xenopus laevis p53
protein (Xp53) in the amphibian cells is initially
found within the cytoplasm. In mammalian cells,
the newly synthesised p53 in the cytoplasm is
activated by phosphorylation and transported
into the nucleus, where it will be able to bind with
DNA, thus initiating transcription of an array of
genes that expand the functionality of the p53.
The same phosphorylated activation process of
Xp53 will occur in X. laevis cells (3). In mammalian cells, p53 can be inhibited as a consequence of being bound to Mdm2. In Xenopus cells,
its equivalent (Xdm2) appears to function similarly. The binding to Mdm2 can inactivate p53,
either by preventing it from binding DNA or, as
Mdm2 has E3 ubiquitin ligase activity, the binding may lead to the degradation of p53 (4).
Depending on the nature of the stress affecting
mammalian cells, Mdm2 is able to increase p53
activity by binding p53-mRNA, thus enhancing its
translation (5). A complex of p53-mRNA–Mdm2
can also regulate the activation and nuclear
translocation of p53 following DNA damage (6).
The enhanced translation of p53-mRNA leads to
cell arrest or apoptosis, in accordance with the
extent of the damage inflicted.
Constitutive Xp53 may play a role in protection
against cancer development in amphibians, as its
constant presence could signal the removal of
altered or damaged cells by apoptosis, rather than
allowing them to transform into cancer cells.
Cancer in amphibians is rare, and additionally, it
is very difficult to induce by using methods that
are effective in mammals (7).
DNA damage and oncogenic deregulation can
stimulate the transmission of a death signal
directly from p53 to the mitochondria, affecting the
permeability of the mitochondrial membrane and
the release of an array of factors relevant to cell
death, e.g. cytochrome c, into the cytosol (8).
In responding to DNA damage, p53 will activate a
variety of gene functions that affect cell cycle arrest,
apoptosis, and senescence, all three of which are
capable of playing a role in tumor suppression. Here,
we will focus on apoptosis. Despite serving as a transcription factor, p53 typically induces apoptosis
through a transcription-independent pathway (9).
Pro-apoptotic gene products are additionally
induced selectively by p53 in response to cellular
stress. However, their activities are thought to be
insufficient to explain the full range of apoptotic
activities subsequently initiated (8). The mitochondrial or intrinsic apoptotic pathway appears to be
the critical apoptotic pathway used in mammalian
cells. It is initiated by a direct effect of activated-p53
acting on the mitochondria (9).
In a recent Comment (10), it was suggested that
“direct-apoptosis” might play an important role in
amphibian apoptosis during their dramatic metamorphosis, as well as in reducing susceptibility to
cancer. In that report, “direct-apoptosis” referred to