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MARÍA ÁNGELES GARCÍA Y COLS. AN. R. ACAD. NAC. FARM.

(30 days post-inoculation), mice were injected by intraperitoneal route
with 5x107 pfu of vaccinia virus VVPKR, VVLUC, VVK296R, or saline
PBS. Virus inoculation by i.p was performed twice in animals with
developed tumours, giving the virus at times 0 and after 3 days.
Tumours were measured every 3-4 days using digital calliper.

3. RESULTS

3.1. VACV recombinants with PKR under control of the lac
I operator/repressor system induced PKR in tumour
prostate cancer cells

We have previously described a VACV recombinant expressing PKR
(VV-PKR) under control of the inducible lac I operator/repressor sys-
tem (9, 11); scheme in Figure 1A). Briefly, when cultured cells are in-
fected with VV-PKR in the presence but not in the absence of IPTG,
PKR is produced at high levels and this leads to its own phosphory-
lation triggered by the viral dsRNA produced during infection from
symmetrical transcription, followed by phosphorylation of eIF-2 al-
pha and in turn, inhibition of protein synthesis, activation of caspas-
es, induction of apoptosis and cell death (6, 12). However, we noted
that when tumour cells TRAMP-C1 are infected with VV-PKR in the
absence of IPTG there low levels of PKR are produced when com-
pared to the infection in the presence of IPTG (Figure 1B). Similar
observation was found when TRAMP-C1 cells were infected with the
mutant form of PKR (K296R), in which case the presence of IPTG
markedly increased the levels of PKR (Figure 1B). As expected, high
levels of PKR produced in the presence of IPTG correlated with phos-
phorylation of eIF-2a, while low levels of PKR in the absence of IPTG
induced lower eIF2a phosphorylation. This was not observed when
the catalitically inactive mutant form of PKR (K296R) was synthe-
sized (Figure 1B). As determined by Western blot with antibodies
against vaccinia proteins, the accumulation of viral proteins was not
impaired in TRAMP-C1 cells infected with VV-PKR in the absence of
IPTG, while it was blocked when IPTG was present (Figure 1C).
Moreover, virus yields were similar in cells infected with the control
virus VV-LUC, a virus expressing luciferase marker in the TK locus,
as in cells infected with VV-PKR in the absence of IPTG (Figure 1D),

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