The EMBO Journal vol.8 no.12 pp.3685-3691, 1989
Proliferation of human malignant melanomas is inhibited
by antisense oligodeoxynucleotides targeted against
basic fibroblast growth factor
Dorothea Becker, Carla B.Meier and
Meenhard Herlyn1
Department of Tumor Biology, M.D. Anderson Cancer Center,
Houston, TX 77030 and 'The Wistar Institute, 36th Street at Spruce,
Philadelphia, PA 19104, USA
Communicated by W.Doefler
Human malignant melanomas, unlike normal melanocytes, can proliferate in the absence of exogenous basic
fibroblast growth factor (bFGY). Exposure of primary
melanomas in the vertical growth phase and metastatic
melanomas to antisense oligodeoxynucleotides targeted
against three different sites of human bFGF mRNA
inhibited cell proliferation and colony formation in softagar. In contrast, exposure of human bFGF sense or
antisense oligonucleotides complementary to human (3nerve growth factor or insulin-like growth factor I mRNA
had no such effects. These experiments indicate that
activation of the bFGF gene may play an important role
in the progression from melanocytic precursor lesions to
malignant melanoma.
Key words: antisense oligodeoxynucleotides/basic fibroblast
growth factor/human malignant melanoma
Introduction
Approximately 95% of familial malignant melanomas and
40 % of sporadic melanomas arise from precursor lesions
(Clark et al., 1984, 1986; Clark, 1988). The three types of
melanocytic lesions are: congenital, common acquired and
dysplastic nevi. Congenital and common acquired nevi
represent focal proliferations of normal human melanocytes.
In contrast, dysplastic nevi consist of a heterogeneous
population of normal melanocytes and melanocytes showing
increased pigmentation, nuclear pleomorphism and mitotic
atypia (Greene et al., 1985). For this reason, melanocytes
of dysplastic nevus are considered to represent precursor
lesions of human malignant melanoma. Human melanoma
can be classified into three stages: i) melanoma in the radial
growth phase, ii) melanoma in the vertical growth phase and
iii) metastatic melanoma. Primary melanoma in the radial
and vertical growth phase and metastatic melanoma
demonstrate significant biological, biochemical and
karyotypic differences from normal human melanocytes and
from melanocytic precursor lesions. For example, primary
melanoma in the vertical growth phase and metastatic
melanomas, unlike normal melanocytes, do not require the
presence of phorbol esters for proliferation in vitro, display
anchorage-independent growth in vitro, form tumors in nude
mice (Herlyn et al., 1985, 1987) and demonstrate severe
karyotypic abnormalities (Balaban et al., 1984, 1986). Most
importantly, however, metastatic melanoma cells can grow
in protein-free medium (Rodeck et al., 1987) whereas
©IRL Press
normal melanocytes require exogenous basic fibroblast
growth factor (bFGF) for proliferation in vitro (Halaban
et al., 1987, 1988a; Herlyn et al., 1988), suggesting that
tumor progression in the melanocytic system is characterized
by increasing independence from exogenous bFGF.
Human bFGF cDNA clones were first isolated and
characterized by Abraham et al. (1986a). The coding region
of the human bFGF gene is interrupted by two introns and
the estimated size of the gene represents at least 34 kb
(Abraham et al., 1986a). In the case of primary human
melanomas in the vertical growth phase and metastatic
melanomas, three bFGF-specific mRNA transcripts of 7.0,
3.7 and 1.2 kb were detected (H.Menssen, U.Rodeck,
L.Otwos, M.L.Mancianti and M.Herlyn, manuscript
submitted). bFGF shares 55% homology with acidic FGF
(Abrahams et al., 1986a) and between 40 and 50%
homology with oncogene hst(K-fgf) (Sakamoto et al., 1986;
Taira et al., 1987; Delli-Bovi and Basilico, 1987; Delli-Bovi
et al., 1987, 1988), the FGF-S gene (Zhan et al., 1987,
1988) and the int-2 gene (Moore et al., 1986). bFGF is
synthesized as a 155 amino acid protein (Abraham et al.,
1986a), which as recently shown by Feige and Baird (1989),
can serve as a substrate for protein phosphorylation.
To determine the role of bFGF in regulating melanoma
development, we studied the effect of antisense oligodeoxynucleotides complementary to three different regions
of human bFGF mRNA on primary melanomas in the
vertical growth phase and on metastatic melanomas. In an
attempt to bypass cell surface-mediated events and directly
influence the expression of a given gene of interest,
application of antisense oligodeoxynucleotides has proved
to represent a powerful tool. For example, Zamecnik et al.
(1986) and Goodchild et al. (1988) demonstrated inhibition
of HIV replication upon addition of antisense oligomers
targeted against viral mRNA. Studies by Wickstrom et al.
(1988) and Holt et al. (1988) demonstrated inhibition of
HL60 cell proliferation and c-myc expression following
addition of antisense oligomers directed against human cmyc mRNA. Oligomer complementary to c-myb-encoded
mRNA was recently shown to inhibit normal human
hematopoiesis in vitro (Gewirtz and Calabretta, 1988) and
proliferation of human myeloid leukemia cell lines (Anfossi
et al., 1989).
In the present study, we demonstrate inhibition of
proliferation of primary human melanoma in the vertical
growth phase and metastatic melanoma upon addition of
antisense oligodeoxynucleotides complementary to the
translation start site and two different splice donor-acceptor
sites of human bFGF.
Results and Discussion
Unmodified, 15 bp oligodeoxynucleotides complementary
to three sites of the sense or antisense strands of human
bFGF mRNA were synthesized. The first l5mer antisense
3685
D.Becker, C.B.Meier and M.Herlyn
A
WM75 (vertical growth phase)
i
=
1;
P"
11
,'.1-
--
days
B
.,~ ~ ~
~~~~~.L
Fig. 1. (A) Kinetics of bFGF-specific oligomer uptake. 32P 5' end
labeled losplice-junction-specific antisense oligomer (5'-TAG CTT
GAT GTG AGG-3') was added to WM852 metastatic melanoma cells
and incubated with the cells for 1, 6, 24 and 36 h. Oligomer uptake
was determined by liquid scintillation counting of the supernatant
media -A- and the aqueous phases of the cell extracts -A-. (B)
Stability of bFGF-specific losplice-junction oligomer upon incubation
with WM852 melanoma cells. Aliquots representing equal numbers of
c.p.m. of the supernatant media and the aqueous phases of the cell
extracts at 1, 6, 24 and 36 h were lyophilized and electrophoresed in a
20% denaturing polyacrylamide gel. The size of DNA fragments
comigrating with xylene cyanol (x) and bromophenol blue (b) in a
20% denaturing polyacrylamide gel are -28 and 8 nucleotides
respectively.
oligomer (5'-GGC TGC CAT GGT CCC-3') was directed
against the translation start site (AUG codon) (Abraham
et al., 1986a) and surrounding nucleotides. The second
antisense oligomer (5'-TAG CTT GAT GTG AGG-3') was
complementary to codon 60, the first splice donor -acceptor
site (Abraham et al., 1986a), and is referred to as losplicejunction oligomer. The third antisense oligomer (5'-ACA
TTT AGA AGC CAG-3') was targeted against codons 94
and 95, representing the second splice donor -acceptor site
(Abraham et al., 1986a) (20splice-junction oligomer).
Although the human bFGF gene shares -50% homology
with acidic FGF (Abraham et al., 1986a) and the genes
hst(K-fgfl (Sakamoto et al., 1986; Taira et al., 1987; DelliBovi and Basilico, 1987; Delli-Bovi et al., 1987, 1988),
FGF-S (Zhan et al., 1987, 1988) and int-2 (Moore et al.,
1986), the sequence of the three different human bFGF
specific antisense oligomers was sufficiently divergent from
the sequence of each of these genes to prevent duplex
formation with their respective mRNAs.
The four cell lines used in the present study were isolated
from melanomas WM75 (primary melanoma in the vertical
growth phase) (Herlyn et al., 1985), WM983-A (primary
melanoma in the vertical growth phase) (R.Kath, U.Rodeck,
A.Parmiter, J.Jambrosie and M.Herlyn, manuscript
submitted), WM983-B, a metastatic melanoma removed
from the same patient as melanoma WM983-A (Kath et al.,
manuscript submitted), and a second human metastatic
melanoma (WM852) (Herlyn et al., 1985).
Northern blot analysis demonstrated bFGF gene
expression in these cell lines, as indicated by the presence
3686
days
c
0
2
*
e
*o
days
Fig. 2. Inhibition of melanoma cell proliferation upon addition of
bFGF-specific antisense oligomers. (A) WM75 cells were plated at a
density of 104 cells/microtiter well in medium containing 5% serum.
After 24 h, the medium was changed and sense and antisense
oligomers were added at 50 ,M/microtiter well. Cell counts were
performed over 8 days, with two replicate cultures for each time
point. Symbols: -0- no oligomer added; -O - sense oligomer, AUG
codon (5'-GGG ACC ATG GCA GCC-3');
antisense oligomer,
losplice-junction (5'-TAG CTT GAT GTG AGG-3'). (B) WM983-B
cells were plated, and oligomers were added as described in (A).
Symbols: -U- sense oligomer, 20splice-junction (5'-CTG GCT TCT
AAA TGT-3'); -O- antisense oligomer, 20splicejunction (5'-ACA
TTT AGA AGC CAG-3'). (C) WM852 cells were plated at a density
of 105 cells/microtiter well and incubated in protein-free medium.
Oligomers were added at 50 tM/microtiter well 24 h later. Symbols:
-M- sense oligomer, losplice-junction (5'-CGT CAC ATC AAG
CTA-3'); -EO- antisense oligomer, losplice-junction; -A- antisense
oligomer, AUG codon (5'-GGC TGC CAT GGT CCC-3').
-U-
of 7.0, 3.7 and 1.2 kb mRNA transcripts (Menssen et al.,
manuscript submitted) detected upon hybridization of the
melanoma mRNAs to a 1.4 kb bovine bFGF-specific cDNA
probe (Abraham et al., 1986b). In contrast, Halaban et al.
bFGF and the development of human malignant melanoma
WM983-B (nvetastatic growth phase)
-
2 xOe
U
days
Fig. 3. Dose -response curve of bFGF-specific antisense oligomer.
WM983-B cells were plated at a density of 104 cells/microtiter plate.
After 24 h 10 -0-, 25 ---, 50 -A- and 75 -U- ltM of losplicejunction-specific antisense oligomer was added to the cells with two
replicate cultures for each time point. Control cultures -0- did not
receive oligomer.
(1988b) demonstrated that bFGF mRNA transcripts and
bFGF immunoprecipitable protein cannot be detected in
normal human melanocytes suggesting that the lack of bFGF
production is at the level of transcription.
Kinetics of bFGF-specific oligomer uptake by human
melanoma cells
To first determine whether oligomers complementary to
bFGF mRNA were efficiently taken up by melanoma cells
in vitro, purified 5' end labeled antisense oligomer directed
against the losplice-junction was added to WM983-A and
WM852 cells (1.2 x 106 c.p.m./106 cells/60 mm plate)
grown in 5% fetal bovine serum, heat inactivated at 65°C
to destroy nuclease activity. Oligomer uptake was determined
by liquid scintillation counting of the supematant media and
the cell extract aqueous phases over 36 h. Figure IA shows
a representative example of the kinetics of oligomer uptake.
Little antisense oligomer was associated with individual
extracts of WM852 cells within 24 h of its addition. More
than 30% of the initial radioactivity added to the cells was
detected between 24 and 36 h in the cell extract aqueous
phases suggesting that the uptake of oligomers by human
melanoma cells represents a rather slow process. To
determine bFGF-specific oligomer stability equal numbers
of c.p.m. (5 x 103 c.p.m./lane) representing the
supernatant media and the aqueous phases representing the
cell pellets were lyophilized and electrophoresed in a 20%
denaturing gel. As shown in an autoradiograph depicted in
Figure 1B, the 5' end labeled bFGF-specific antisense
oligomer was not degraded even after 36 h of incubation
with WM852 cells. The finding that oligomers are stable
for a significant period of time upon incubation with cells
was also reported by Holt et al. (1988) who observed the
presence of full-length c-myc oligomer 5 days after addition
to the medium of HL60 cells.
24 h, at which point the cells had reached 30-40%
confluency, single doses of 50 AtM bFGF-specific sense or
antisense oligomers were added. On days 2, 4, 6 and 8
following addition of the oligomers, the cells were
trypsinized, resuspended in 1.00 ml of medium, assayed for
viability by trypan blue exclusion and counted. Regardless
of whether incubation took place in the presence of sense
or antisense oligomers, the cells demonstrated 98-100%
viability and no signs of morphological changes. Furthermore, in neither of the experiments described here, did the
sense, antisense or untreated melanoma cells reach
confluency on day 8 of the incubation, thereby ruling out
the possibility that the results obtained were due to having
measured confluency rather than antisense oligomer induced
inhibition of cell proliferation.
As shown in Figure 2A, WM75 cells grown in the
presence of sense oligomer specific for the initiation codon
(5'-GGG ACC ATG GCA GCC-3') grew from l04 to
106 cells/ml which was comparable to the number of
cells counted on day 8 which had not received oligomers.
In comparison, WM75 cells grown in the presence of bFGFspecific antisense oligomer complementary to the losplicejunction, revealed -60% growth inhibition measured on day
8. Similar results (data not shown) were obtained in case
of WM983-A, a primary melanoma in the vertical growth
phase.
Figure 2B demonstrates the growth inhibitory effects of
50 ItM 20splice-junction-specific antisense oligomer on the
metastatic melanoma cell line, WM983-B. By days 6 and
8, the extent of growth inhibition in the presence of antisense
compared with sense oligomer (5'-CTG GCT TCT AAA
TGT-3') represented 50-70%. Addition of the same
20splice-junction-specific sense and antisense oligomers to
WM983-A cells resulted in a similar extent of growth
inhibition (data not shown).
Since it is one of the characteristics of metastatic melanoma
cells to proliferate in the absence of serum without
demonstrating any signs of morphological changes (Rodeck
et al., 1987), WM852 and WM983-B cells were adapted
to growth in protein-free medium over a period of 5 weeks.
At the end of 5 weeks, WM852 and WM983-B were plated
at a density of 105 cells/microtiter well followed 24 h later
by the addition of 50 AM losplicejunction-specific sense
oligomer (5'-CCT CAC ATC AAG CTA-3') or 50 AM of
each of the three bFGF-specific antisense oligomers. The
results demonstrated between 50 and 75% inhibition of
proliferation of WM852 (Figure 2C) and WM983-B cells
(data not shown) in the presence of each of the three antisense
oligomers compared with the 1°-splice-junction-specific
sense oligomer.
The dose of 50 AtM of bFGF-specific antisense oligomers
was chosen for the studies presented here based upon the
result of a dose -response curve (Figure 3), which revealed
that incubation of WM983-B cells in the presence of 10 AtM
losplice-junction-specific antisense oligomer compared with
50 ItM losplice-junction-specific antisense oligomer showed
50% less growth inhibition. On the other hand, addition
of 75 AM in comparison with 50 AtM bFGF-specific
losplice-junction antisense oligomer did not demonstrate a
significantly higher degree of inhibition of proliferation.
The extent of growth inhibition of 50-75% upon addition
of bFGF-specific antisense oligomers to human malignant
melanomas, compared to untreated or bFGF-specific sense
_
-
Oligomers complementary to human bFGF are growth
inhibitory to human malignant melanomas
To assess the effects of bFGF-specific sense and antisense
oligomers, WM75 and WM983-B cells were plated in
medium containing 5 % fetal bovine serum at a density of
I04 cells/microtiter well in 24 well microtiter plates. After
3687
D.Becker, C.B.Meier and M.Herlyn
A
WM983-B (metastatic growth phase)
WM953-B (meailc growth phk)
I
days
B
WM983.B({*statk
days
growth phas)
Fig. 5. bFGF-specific antisense oligomer induced inhibition of
melanoma cell proliferation demonstrates sequence specificity.
Oligomers were added at 50 uM/microtiter well to WM983-B cells
grown in medium containing 5% serum. The growth curves were
performed as described in Figure 2, with replicate cultures for each
time point. Symbols: -O- sense oligomer directed against the losplicejunction (5'-CCT CAC ATC AAG CTA-3'); -A- 5 bp mismatched
antisense oligomer, losplice-junction (5'-TAG CTT ATG TGG AGG3'); -OII- antisense oligomer, losplice-junction (5'-TAG CTT GAT
GTG AGG-3').
I
'-
days
days
Fig. 4. (A) Inhibition of proliferation of human melanomas by bFGFspecific antisense oligomers is reversible. Replicate cultures of
WM983-B cells (104 cells/microtiter well) were incubated in the -Oabsence of oligomer or in the -i- presence of 50 /%M losplicejunction-specific oligomer for 8 days. On day 8, the two melanoma
cell cultures were rinsed three times with medium and incubated for an
additional period of 48 h in the presence of medium containing 5%
serum but no oligomer. Growth curves were established as described
in Figure 2. (B) Two sets of replicate cultures of WM983-B cells
(104 cells/microtiter well) were cultured for 8 days in the presence of
50 AtM bFGF-specific antisense oligomer targeted aainst the losplice-. On day 8, 3-fold excess (150 ItM) 1 splice-junctionjunction
specific sense oligomer was added to half of the cell cultures -0-,
whereas the other half did not receive additional oligomer *-.
WM983-B cells were cultured for another 2 days and growth curves
were established as described. (C) Exogenous bovine bFGF functions
as a 'competitor' for antisense oligomer-induced inhibition of
proliferation. WM852 cells adapted to growth in protein-free medium
were plated at a density of 5 x 104 cells/microtiter well and incubated
for 8 days in the absence of oligomer -C -, in the presence of
40 ng/ml of bovine bFGF, in the presence of 50 ltM antisense
oligomer directed against the losplice-junction -0-, or in the presence
of 50 AM losplice-junction-specific antisense oligomer and 40 ng/ml of
bovine bFGF ---.
-.
-
-.
-
oligomer treated melanoma cells, was similar to the decrease
in the growth rate reported for HL60 cells upon incubation
with c-myc antisense oligomer (Holt et al., 1988) or upon
addition of c-myb antisense oligomer (Anfossi et al., 1989).
3688
Antisense oligomer effects on melanoma cell
proliferation are reversible
Assuming that the mechanism responsible for the observed
inhibition of proliferation of human malignant melanomas
is due to duplex formation between intracellular bFGF
mRNA and the corresponding antisense oligomers, one
would predict that antisense oligomer effects are reversible.
Two independent experiments were designed to provide
experimental evidence for this hypothesis.
WM983-B cells were incubated in the gresence of 50 4M
antisense oligomer targeted against the 1 splice-junction for
8 days, which led to 70% growth inhibition compared to
untreated WM983-B cells (Figure 4A). On day 8, the
medium was either replaced with fresh medium containing
no oligomer (Figure 4A) or as a separate experiment
(Figure 4B), 3-fold (150 ,1M) excess of losplicejunctionspecific sense oligomer was added to the cells without a
medium change. Replacement with medium containing no
oligomer, as well as hybridization competition with sense
oligomer led to complete reversal of growth inhibition within
48 h (Figure 4A and B). Reversible antisense oligomer
induced inhibition of proliferation has also been demonstrated
for HL60 cells incubated in the presence of c-myc specific
antisense oligomers (Holt et al., 1988) and for BALB/c3T3
cells incubated in the presence of antisense oligomers
complementary to PCNA, a nuclear protein representing a
cofactor of DNA polymerase 6 (Jaskulski et al., 1988).
Given the context of the present study, another important
aspect to investigate was the possibility that bFGF-specific
antisense oligomer induced inhibition of proliferation of
human melanomas would be abolished in the presence of
exogenous bFGF. To test this hypothesis, WM852 cells
adapted to growth in protein-free medium were plated at a
density of 5 x 104 cells/microtiter well. After 24 h, the
cells received either no oligomer, no oligomer but 40 ng/ml
exogenous bovine bFGF, 50 ItM bFGF-specific antisense
oligomer directed against the losplice-junction or the same
antisense oligomer together with 40 ng/ml of bovine bFGF.
bFGF and the development of human malignant melanoma
WM9W3-B (elasatic growth phae)
2.10
Table I. Inhibition of anchorage-independent growth by antisense
oligomers directed against human bFGF mRNA
WM983-B (metastatic growth phase)
Cell type
(5 x 103)
i.
d'.0
2
I-
4
C
a
Oligodeoxynucleotide
25 AM
Number
of soft agar colonies
counted on day 14
Dish
1
2
mean
35
28
8
4
4
27
13
2
5
4
31
21
5
5
4
'O0
days
Fig. 6. Antisense oligomers targeted against human ,3-NGF and IGF-I
mRNA have little growth inhibitory effect on melanoma cell
proliferation. Oligomers were added at 50 uM/microtiter well to
WM983-B cells grown in medium containing 5% serum. The growth
curves were performed as described in Figure 2. Symbols: -O - no
oligomer; -0- antisense oligomer directed against human IGF-I
mRNA (5'-CAT CTT CAC CTT CAA-3'); -A- antisense oligomer
complementary to human ,B-NGF mRNA (5'-ATG CAC CTC ACT
GCG-3'); -]- bFGF-specific antisense oligomer targeted against the
losplice-junction.
The data presented in Figure 4C revealed two interesting
findings. Firstly, the growth rate of WM852 cells incubated
in the presence of exogenous bFGF was not significantly
elevated compared with the growth rate of WM852 cells
cultured in protein-free medium, thus supporting the
hypothesis that human malignant melanomas synthesize
sufficient quantitities of bFGF required for their proliferation.
Secondly, WM852 cells grown in the presence of bFGFspecific antisense oligomer and exogenous bFGF reached
similar cell densities on days 2, 4 and 6 as untreated WM852
cells, thus indicating that exogenous bFGF functioned as a
'competitor' for antisense oligomer-inducible inhibition of
proliferation.
'Mismatched' bFGF antisense oligomer and antisense
oligomers unrelated to bFGF have few growth
inhibitory effects
To address the aspect of the specificity of inhibition of the
human bFGF gene, a l5mer antisense oligomer complementary to the losplice-junction with ten identical and five
mismatched base compositions (5'-TAG CTT ATG TGG
AGG-3') produced no apparent inhibition of cellular growth
when added to WM983-B metastatic melanoma cells at a
concentration of 50 AM (Figure 5), thus indicating that the
specific hybrid formation between bFGF-specific antisense
oligomers and endogenous bFGF mRNA was responsible
for the inhibition.
Ross et al. (1984) demonstrated the expression of nerve
growth factor receptor on melanoma and dysplastic nevus
cells, as Peacocke et al. (1988) did on normal human
melanocytes upon the addition of the tumor promoter 12-0tetradecanoyl phobol-13-acetate (TPA) to the culture
medium. Furthermore, recent investigations (Rodeck et al.,
1987) indicated that insulin-like growth factor I (IGF-I) and
insulin represent major growth factors for melanomas. To
determine whether the synthesis of human 3-nerve growth
factor (f-NGF) and/or IGF-I by human malignant
melanomas is as important for their proliferation as the
production of bFGF,. l5mer antisense oligomers targeted
against the splice donor-acceptor site of human f-NGF
WM983-B
WM983-B
WM983-B
WM983-B
WM983-B
sense losplice-junction
antisense AUG codon
antisense losplice-junction
antisense 20splice-junction
Soft agar colony forming efficiency of human melanomas in the
presence of bFGF-specific oligomers. 5 x 103 WM983-B melanoma
cells were resuspended in 0.3% soft agar medium containing 5%
serum, and either no oligomers or 25 itM of either sense oligomer
specific to the losplice-junction or antisense oligomers complementary
to the AUG codon or to the 10 or 20splice-junction were added. The
cells were plated into 35 mm Petri dishes and individual soft agar
colonies were counted on day 14 following addition of the different
oligomers. The data presented here represent one of three soft agar
assays performed, all of which led to similar results.
(residues -166/ - 125) (5'-ATG CAC CTC ACT GCG-3')
(Ullrich et al., 1983) and human IGF-I (first exon-intron)
(5'-CAT CTT CAC CTT CAA-3') (Bell et al., 1985) were
added to WM983-B cells. As compared with untreated cells,
no significant growth inhibition was observed in the presence
of either of the two bFGF-unrelated antisense oligomers over
a period of 8 days (Figure 6). On the other hand, the
l1splice-junction bFGF-specific antisense oligomer inhibited
the proliferation of WM983-B cells by 70% in comparison
with WM983-B cells which did not receive oligomers
(Figure 6). These results therefore suggest that the
proliferation of human malignant melanomas is particularly
dependent upon the autocrine production of bFGF rather than
the synthesis of human ,B-NGF or IGF-I.
Inhibition of anchorage-independent growth of human
malignant melanomas upon addition of bFGF-specific
antisense oligomers
Since the initial experiments described here suggested that
the synthesis of bFGF was important to the autocrine growth
activities of human malignant melanomas, we investigated
whether bFGF-specific antisense oligomers also inhibited the
ability of melanoma cells to form colonies in soft agar. The
results, as shown in Table I, revealed 40% fewer soft agar
colonies in the presence of 25 /M of each of the three
antisense oligomers compared with AUG codon-specific
sense oligomer-treated cells and 75 % less colony formation
than seen in untreated cells.
The data presented here demonstrate that proliferation of
primary human melanomas in the vertical growth phase and
metastatic melanomas is particularly dependent upon the
autocrine production of bFGF. This finding, together with
the fact that normal human melanocytes cannot proliferate
in the absence of exogenous bFGF (Halaban et al., 1987,
1988a; Herlyn et al., 1988), suggest that bFGF gene
activation may represent an important step in the
3689
D.Becker, C.B.Meier and M.Herlyn
development of human melanomas, possibly correlating with
the onset of melanoma progression through the epidermis - dermis in a vertical fashion.
The possibility that autocrine production of bFGF
represents an event which may contribute to tumor
progression was also suggested by Schweigerer et al. (1987),
who demonstrated that human embryonal rhabdomyosarcomas, unlike normal myoblasts (Gospodarowicz et al.,
1976), produce bFGF which stimulates their own growth
and that of vascular endothelial cells.
Finally, as shown in this study, application of antisense
oligodeoxynucleotides has proven to represent an excellent
approach to determine the importance of specific gene(s)
involved in the progression of human malignant melanoma
and its precursor lesions.
0.3% soft agar medium containing 5% serum and 25 /tM of oligomer. The
cells were plated into 35 mm Petri dishes and soft agar colonies were counted
after 14 days of incubation.
Acknowledgements
We wish to thank G.Lozano and J.H.Chen for critical comments on the
manuscript. Oligonucleotides were synthesized by the Macromolecular
Synthesis Facility of the M.D.Anderson Cancer Center supported by
CA16672. M.H. was supported by NIH grant CA25874.
References
Abraham,J.A., Whang,J.L., Tumolo,A., Mergia,A., Friedman,J.,
Hjerrild,K.A., Gospodarowicz,D. and Fiddes,J.C. (1986a) EMBO J.,
5, 2523-2528.
Abraham,J.A., Mergia,A., Whang,J.L., Tumolo,A., Friedman,J.,
Hjerrild,K.A., Gospodarowicz,D. and Fiddes,J.C. (1986b) Science, 233,
545-548.
Materials and methods
Synthesis and purification of oligomers
Unmodified, l5mer oligodeoxynucleotides were synthesized on a 1 itM scale
on an Applied Biosystems 380B DNA synthesizer by means of 3-cyanoethyl
phosphoramidite chemistry. The oligomers were purified by HPLC,
lyophilized, resuspended in 0.1 M NaCI and purified further on ion exchange
resin columns (NACS Prepac, BRL).
Cell lines
The primary melanoma cell lines in the vertical growth phase WM75 and
WM983-A, and the metastatic melanoma cell lines WM983-B and WM852
employed in this study, have been described (Herlyn et al., 1985; Kath
et al., manuscript submitted).
The cells were maintained in three parts Temins's modified MEM and
one part Leibovitz's L15 medium supplemented with 5% heat inactivated
(56°C for 30 min) fetal bovine serum at 35°C in 5% CO2. WM852 and
WM983-B melanoma cells adapted to growth in protein-free medium were
cultured in three parts Temin's modified MEM and one part Leibovitz's
L15 medium over a period of 5 weeks by plating the cells at a minimal
density of 3 x 104 cells/cm2.
Experiments involving oligomers were performed with a single lot of fetal
bovine serum which had been heat inactivated at 65°C for 45 min to destroy
nuclease activity.
Determination of oligomer uptake and stability
1°splicejunction specific antisense oligomer was 5' end labeled with
[-y- 2P]ATP (3000 Ci/mmol; NEN) with T4 polynucleotide kinase and
purified on a NACS Prepac column or a 20% denaturing polyacrylamide
gel. 1.2 x 106 c.p.m. of 5' end labeled antisense oligomer was added to
WM852 cells which had been plated at a density of 106 cells/60 mm plate
in medium containing 5% serum 24 h prior to addition of the oligomer.
Following addition of the oligomer samples were incubated for 1, 6, 24
and 36 h. Supernatant media (1.00 mil/plate) were removed at each time
point and centrifuged to remove cellular debris (Wickstrom et al., 1988).
The cell pellets were rinsed twice with phosphate-buffer saline (PBS), lysed
in 1.00 ml of Tris-buffered saline containing 1% SDS (Wickstrom et al.,
1988) and extracted with phenol -SSC. Uptake of antisense oligomer at
each time point was determined by liquid scintillation counting of aliquots
of the supernatant media, the PBS washes and the aqueous phases of the
cell pellets. Oligomer stability was determined by analysis of lyophilized
aliquots (5 x 103 c.p.m./sample) of the supematant media and the aqueous
phases of the cell pellets in a 20% denaturing polyacrylamide gel. The
lyophilized aliquots were resuspended in formamide buffer containing
bromophenol blue and xylene cyanol which served as size markers.
Determination of growth rates
Melanoma cells were plated at a density of 104 cells/microtiter well in
24 well plates or at 10 or 5 x I04 cells/microtiter well when grown in
protein-free medium. After 24 h the cells were rinsed twice with medium
and fresh medium containing sense or antisense oligomers was added
(0.25 ml medium/microtiter well). Cell counts were determined in duplicate
samples at each time point in each of the experiments. Viability of the cells
was analyzed by trypan blue exclusion.
S x 103 WM983-B cells were resuspended in a final concentration of
3690
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Received on 31 July, 1989; revised on 5 September, 1989
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