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

zyme that when activated by dsRNA is authoposphorylated leading to
phosphorylation of the alpha subunit of the eukaryotic initiation fac-
tor eIF-2 (eIF2a) and, in turn, to inhibition of protein synthesis, in-
duction of apoptosis and cell death (for review of PKR see (6)). Since
PKR induction is dependent on sensitivity of the cells to IFN, we rea-
soned that a VACV vector expressing PKR should infect and destroy
better cancer cells than normal cells, as the latter have a fully active
IFN system. Thus, in this investigation we have explored the oncolyt-
ic ability of a VACV recombinant expressing low levels of PKR (re-
ferred to as VV-PKR) in cultured tumour cells and in a mouse mod-
el after subcutaneous inoculation of prostate tumour cells (TRAMP-
C1). The use of a viral recombinant expressing low levels of PKR is
because high levels of expression of PKR prevent virus growth (7, 8).
Our findings showed that VV-PKR replicate in cultured tumour cells,
but when inoculated in subcutaneous TRAMP-C1 tumour-bearing
mice causes reduction of tumour burden in spite of restricted virus
growth.

2. MATERIALS AND METHODS

2.1. Cells and viruses

TRAMP-C1 murine prostate cancer cell line obtained from
American Type Culture Collection was maintained in DMEM with 10%
foetal calf serum (FCS) and antibiotics. Cells reaching 95% conflu-
ence were shortly tripsinized, and harvested with serum-containing
medium. The cells were washed and resuspended in serum-free com-
plete medium at concentration of 5 x 106 viable cells in 1 ml. Half-
million TRAMP-C1 cells in 100 µl were implanted subcutaneously into
the right flank. Tumour volume was estimated using the formula: m12
x m2 x 0.5236, where m1 and m2 represented the short and long diam-
eter of the tumour, respectively. Tumours were measured using a dig-
ital calliper twice weekly until day 55 post-implantation or until the
tumour burden has met the humane endpoint. Tumours that could
not be measured with calliper but were found to infiltrate surround-
ing tissues at the necropsy were assigned as immeasurable. Tumour-
free status was assigned to mice that at the necropsy have shown lack
of residual tumour tissue at the implantation site.

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