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Survivin is a structurally unique mem-
ber of the inhibitor of apoptosis (IAP)
family of proteins that is potentially
involved in both control of cell divi-
sion and inhibition of apoptosis (1).
Specifically, its antiapoptotic function
is related to the ability to directly or
indirectly inhibit caspases (2). The
notion that survivin is overexpressed
in most of the common human
tumors (3, 4) but absent in normal
adult tissues with only a few excep-
tions (5, 6) has led to the proposal of
survivin as a promising therapeutic
target for novel anticancer therapies
(7). Indeed, in October 2001, Mesri et
al. (8) reported in the JCI that infec-
tion with a replication-deficient aden-
ovirus encoding a Thr34→Ala mutant
of survivin caused apoptosis in
human tumor cell lines of different
histology and reduced tumor growth
in xenograft breast cancer models.
Moreover, inhibition of survivin
expression enhanced taxol-induced
cell death in tumor cells.
As an alternative strategy for survivin
inhibition we developed hammerhead
ribozymes targeting the 3′end of the
CUA110 (RZ1) and the GUC294 (RZ7)
triplets in the survivin mRNA. In a cell-
free system, both ribozymes induced
cleavage of a synthetic RNA substrate
obtained by cloning a portion of sur-
vivin mRNA, with cleavage products
being detectable starting from a
ribozyme/substrate ratio of 1:0.5. Con-
versely, the catalytically inactive
mutRZ1 (which was produced by intro-
ducing a mutation in the catalytic core
of the active ribozyme RZ1 and was
used as control throughout the study)
did not show any cleavage activity. RZ1,
RZ7, and mutRZ1 sequences were
inserted into the pRC expression vector
under the control of the cytomega-
lovirus promoter and transfected into
the human metastatic melanoma cell
line JR8 overexpressing survivin. For the
present study we selected three stably
transfected clones proven to endoge-
nously express RZ1 (clone RZ1/C), RZ7
(clone RZ7/H), or mutRZ1 (clone
mutRZ1/B). RZ1/C and RZ7/H cells
were characterized by a markedly lower
survivin protein level (68% and 60%
lower, respectively) than JR8 parental
cells, whereas a negligible reduction
(13%) in survivin expression was
observed in mutRZ1/B cells (Figure 1a).
To evaluate the effect of survivin inhibi-
tion on the susceptibility of melanoma
cells to undergo cisplatin-induced apop-
tosis, we treated the different clones
with 10 µg/ml of the drug for 1 hour
and determined the presence of apop-
totic nuclei in cells stained with propid-
ium iodide under fluorescence micro-
scopy at 72 hours after treatment. A very
modest apoptotic response was ob-
served in the mutRZ1/B cells, whereas a
significant increase in the percentage of
apoptotic cells was observed in RZ1/C
(P = 0.01) and RZ7/H (P = 0.005) cells
(Figure 1b). Processing of caspase-3 to
its active subunits of approximately 17
and 19 kDa was observed in all three
drug-treated clones. However, the cas-
pase-3 catalytic activity as assessed by
hydrolysis of the fluorogenic substrate
N-Acetyl-Asp-Glu-Val-Asp-aldehyde
(Ac-DEVD-AMC) was about threefold
higher in RZ1/C and RZ7/H clones
than in the mutRZ1/B clone (Figure 1c).
These results are in agreement with
the previous finding of Grossman et
al. (9), who observed enhancement of
cisplatin-induced apoptosis by expres-
sion of the survivin Thr34→Ala
mutant in YUSAC2 melanoma cells.
Unlike what was reported by these
authors, attenuation of survivin
expression in our melanoma cell sys-
tem was not sufficient to appreciably
trigger apoptosis in the absence of
other stimuli. Other antiapoptotic
factors besides survivin, such as bcl-2
and bcl-xL, are strongly expressed in
JR8 cells and may contribute to pre-
Ribozyme-mediated attenuation of survivin
expression sensitizes human melanoma cells
to cisplatin-induced apoptosis
The Journal of Clinical Investigation | January 2002 | Volume 109 | Number 2 285
Letters to the Editor
Figure 1
(a) Survivin protein expression in JR8 parental cells and melanoma cell clones transfected with the active ribozymes RZ1 (RZ1/C clone) and RZ7 (RZ7/H
clone) or with the mutant ribozyme mutRZ1 (mutRZ1/B clone). Western blots were probed with a polyclonal antibody for survivin. Proliferating cell
nuclear antigen (PCNA) was used as a control for loading. (band c) Induction of apoptosis in melanoma cell clones exposed to 10 µg/ml cisplatin for
1 hour. Seventy-two hours after treatment, cells were collected and the occurrence of apoptosis was determined. (b) The percentage of cells with an
apoptotic morphology with respect to the overall population was assessed by fluorescence microscopy after cell staining with propidium iodide. White
bars, control (no drug) cells; gray bars, cisplatin-treated cells. Data represent mean values ± SD of three experiments. (c) The caspase-3 catalytic activ-
ity was determined by hydrolysis of the fluorogenic substrate N-Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-AMC) in the presence or absence of the
caspase-3 inhibitor Ac-DEVD-CHO. White bars, control (no drug) cells; gray bars, cisplatin-treated cells; black bars, cisplatin-treated cells + CHO.
Data represent mean values ± SD of three experiments.
In conclusion, the present results
obtained with the ribozyme-mediated
approach in melanoma cells extend
and corroborate earlier evidence indi-
cating that attenuating survivin expres-
sion renders these cells more suscepti-
ble to cisplatin-induced apoptosis.
These data also suggest a possible strat-
egy to enhance the chemosensitivity
profile of such a drug-refractory
human malignancy.
Marzia Pennati,1Gennaro Colella,1
Marco Folini,1Lorenzo Citti,2
Maria Grazia Daidone,1
and Nadia Zaffaroni1
1Department of Experimental Oncology,
Unit 10, Istituto Nazionale per lo Studio
e la Cura dei Tumori, Milan, Italy
2Istituto di Mutagenesi e Differenziamento,
Pisa, Italy
Address correspondence to: Nadia Zaffaroni,
Istituto Nazionale per lo Studio e la Cura dei
Tumori, Via Venezian 1, 20133 Milano, Italy.
Phone: 39-02-23903260; Fax: 39-0223903052;
E-mail: zaffaroni@istitutotumori.mi.it.
Marzia Pennati and Gennaro Colella
contributed equally to this work.
1. Altieri, D.C., and Marchisio, P.C. 1999. Survivin
apoptosis: an interloper between cell death and cell
proliferation in cancer. Lab. Invest. 79:1327–1333.
2. Reed, J.C. 2001. The survivin saga goes in vivo. J. Clin.
Invest. 108:965–969. DOI:10.1172/JCI200114123.
3. Ambrosini, G., Adida, C., and Altieri, D. 1997. A
novel anti-apoptosis gene, survivin, expressed in
cancer and lymphoma. Nat. Med. 3:917–921.
4. Velculescu, V.E., et al. 1999. Analysis of human
transcriptomes. Nat. Genet. 23:387–388.
5. Konno, R., et al. 2000. Expression of survivin and
Bcl-2 in the normal human endometrium. Mol.
Hum. Reprod. 6:529–534.
6. Chiodino, C., et al. 1999. Expression of the novel
inhibitor of apoptosis survivin in normal and
neoplastic skin. J. Invest. Dermatol. 113:415–418.
7.Grossman, D., McNiff, J.M., Li, F., and Altieri,
D.C. 1999. Expression and targeting of the apop-
tosis inhibitor, survivin, in human melanoma. J.
Invest. Dermatol. 113:1076–1081.
8. Mesri, M., Wall, N.R., Li, J., Kim, R.W., and Altieri,
D.C. 2001. Cancer gene therapy using a survivin
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DOI:10.1172/JCI200112983.
9. Grossman, D., Kim, P.J., Schechner, J.S., and
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venting programmed cell death in this
tumor model. However, it should be
stressed that in JR8 cells survivin
expression was attenuated but not
completely abrogated. It may be that
inhibition below a certain threshold is
insufficient to determine a proapop-
totic effect. Interestingly, and in accor-
dance with such a hypothesis, when
we transduced the human prostate
cancer cells DU145 with a Moloney-
based retroviral vector carrying the
catalytic sequence of the ribozyme
RZ7, we were able to select a
ribozyme-expressing clone character-
ized by an almost complete abroga-
tion of survivin expression (99.5%
lower compared with control cells, as
assessed by Western blotting, and lack
of detectable protein expression by
confocal microscopy). This ribozyme-
expressing clone also showed a
markedly higher percentage of apop-
totic cells than control culture (20%
and 3% of cells on the overall cell pop-
ulation, respectively).
286 The Journal of Clinical Investigation | January 2002 | Volume 109 | Number 2
