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a protein classified as tumour suppressor by its capacity to
induce cell cycle arrest and apoptosis in response to a
variety of cellular stresses like DNA damage, transcription
inhibition, depletion of nucleotide pools, oncogene
expression, and heat shock, among others (144). The
induction of p53 by IFN was showed by Takaoka et al. in
2003, and the antiviral activity of this tumour suppressor
was reinforced by the fact that it is frequently targeted by
viral proteins (145). We demonstrated in an elegant way
that VSV is impaired as a result of apoptosis induction via
p53 activation, using a mice model expressing an extra-
copy of p53 called “super p53 mice” (146). Although in
this model we did not directly address the role of PKR in
the antiviral effect of p53, the close link between both
proteins described above suggests great potential for PKR
as a mediator, which should be the subject of other, more
specific studies.
The alternative reading frame (ARF) is one of the two
unrelated products encoded by the INK4a–ARF locus, one
of the most frequently mutated genes in cancer. Initially,
ARF activity was linked to p53 stabilisation after
oncogenic stress, but p53-independent functions have been
also described, placing this tumour suppressor as sensor of
different types of stress (147). The control exerted by the
tumour suppressor Arf on cellular proliferation is crucial to
restrict tumour development. Several reports described the
activation of ARF after the expression of viral proteins,
type I IFN treatment, or after virus infection, suggestive of
a physiological role for ARF during virus infection (1, 2).
However, due to the well-known connection between ARF
and p53, a direct antiviral activity for ARF independently
of p53 activation had not been considered before. Our
results revealed that ARF can be induced by viral infection
and that the expression of ARF reduces viral infectivity. In
fact, ARF is protective against IFN-sensitive viruses such
as VSV, Sindbis virus, or a recombinant VV rendered IFN
sensitive by deletion of the PKR inhibitory gene, E3L. We
have shown that this antiviral effect depends in part, on
PKR activation mediated by its release from inhibitory
complexes with NPM (148). These results provided a new
link between tumour suppression and antiviral host
defence, an important step to understand the tumourigenic
activity of viruses and a crucial learning for the
forthcoming use of viruses as therapeutic agents. Under
the direction of Dr Rivas, and in collaboration with Dr
Esteban and Dr Serrano, these results were published with
my contribution as the first author in the prestigious
EMBO Journal in 2006 (148).
In addition, we suggested other links between tumour
suppression and antiviral host defence, with PKR playing a
role in the activation of the NF-kB pathway through viral
infection. The retinoblastoma protein Rb is a tumour
suppressor involved in cell cycle control, differentiation,
and inhibition of oncogenic properties (149). We showed
that virus replication was increased by the absence of Rb
tumour suppressor gene expression, and that Rb was
required for the activation of the NF-kB pathway in
response to virus infection (150). An analysis of PKR
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María Ángel García Chaves
activation in Rb knockout and wild-type MEFs in response
to VSV infection revealed decreased levels of both
phospho-PKR and phosphor-eI2Fa proteins in the
knockout cells, suggesting PKR as a possible mediator.
These results revealed a novel role for tumour suppressor
Rb in viral infection surveillance and further extend the
concept of a link between tumour suppressors and antiviral
activity. These results were published in 2009, under the
direction of Dr Rivas and Dr Esteban, with my
contribution as first author in Plos One journal (150).
In summary, these data highlight an important role of
different tumour suppressors in the complex innate
antiviral host defence, due in part to the involvement of
PKR activity. While the field of tumour suppressors with
antiviral function is in its infancy, future work will unravel
a wider significance of tumour suppressors in host cell
defence against pathogens. Understanding how tumour
suppressors exert their antiviral function will be relevant in
the potential use of viruses as oncolytic agents and for
gene therapy in cancer, as we have reviewed in depth in
both Carcinogenesis and Future Virology journals (151,
152).
4.1.b. PKR as a molecular target of the 5-fluorouracil
chemotherapeutic drug
The chemotherapeutic drug 5-FU is widely used in the
treatment of a range of cancers, being the first and second
line of treatment in combination in colorectal cancer
patients, and the third line in the palliative care of
numerous cancer types. However, adverse effects and
resistance to the drug remain major clinical problems.
Since defects in the mediators of apoptosis may account
for chemoresistance, the identification of new targets
involved in 5-FU-induced apoptosis is of great clinical
interest. The p53 tumour suppressor has been reported as
an important protein involved in 5-FU-induced apoptosis
(153). However, several works have shown that apoptosis
can also occur in mutant p53 cell lines by a mechanism
still unknown, with p53 playing the role of a biomarker of
response to 5-FU in tumours pending to find new targets
involved in 5-FU sensibility or resistance. We have
identified PKR as a key molecular target of 5-FU involved
in apoptosis induction in human colon and breast cancer
cell lines (154). We analysed PKR distribution and
activation, apoptosis induction, and cytotoxic effects
during 5-FU and 5-FU/IFNa treatment in several colon
and breast cancer cell lines with different p53 status. PKR
protein was activated by 5-FU treatment in a p53-
independent manner, inducing phosphorylation of eIF-2a
and cell death by apoptosis. Furthermore, PKR
interference promoted a decreased response to 5-FU
treatment and those cells were not affected by the
synergistic antitumour activity of 5-FU/IFNa combination.
We have shown that PKR is activated in absence of p53
expression and, whereas PKR knockdown decreased 5-
FU-mediated apoptosis, cell death was completely
abolished in absence of both PKR and p53 proteins. These
results suggest the importance of both proteins in 5-FU-
induced apoptosis, and the relevance acquired by PKR in
@Real Academia Nacional de Farmacia. Spain