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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ARTICLE NO.
225, 808-816 (1996)
1255
Simultaneous Suppression of Progression Marker Genes in the Highly
Malignant Human Melanoma Cell Line BLM after Transfection
with the Adenovirus-5 E1A Gene
Jan J. M. van Groningen,* Ine M. A. H. Cornelissen,f Goos N. P. van Muijen,t
,i
Henri P. J. Bloemers,* and Guido W. M. Swart*
*Department o f Biochemistry and f Department o f Pathology, University o f Nijmegen,
P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
Received July 22, 1996
The highly metastatic human melanoma cell line BLM was transfected with the ElA or EIA+E1B
regions of adenovirus 5 (Ad5). A series of progression markers, correlated with the malignant phenotype
of parental BLM (including calcyclin, thymosin 0 10, plasminogen activator inhibitors types 1 and 2
urokinase type and tissue type plasminogen activators, vimentin, tissue type transglutaminase, and interleu
kin-6), was collectively repressed in the transfeetants, whereas several control genes were not affected or
even induced. The apparently coordinate repression of a set of markers by the same regulator gene, Ad5
ElA in this case, suggests the existence of one pathway under the control of a main switch and predicts
that one or more as yet unidentified cellular master genes normally exert this function. A reduced oncogenic
ity was observed after subcutaneous inoculation of the ElA transfeetants into nude mice and provides
additional evidence in support of a tumor suppressor function of Ad5 E lA . © 1996 Academic Press, inc.
Melanocytic tumor progression is thought to evolve through several distinct stages, from
normal melanocytes to highly invasive melanoma cells capable of metastasis (1,2). A vast
collection of molecular markers associated with subsequent stages of melanocytic tumors and
expressed in a number of melanoma derived cell lines (for review, see ref. 3) has been
described.
In the past years we have characterized several reverse progression markers, present in non
metastatic human melanoma cell lines, as well as positive progression markers, characteristic
for highly metastatic melanoma cell lines (4-10). While changes in gene regulation drive tumor
progression, it should be feasible to identify one or a few regulatory genes (e.g. encoding
transcription factors or coactivators) that control expression of a set of either reverse or positive
progression markers by characterizing the regulatory elements of available marker genes.
Alternatively, it may be possible to test a regulatory gene as a candidate to control the
expression of a set of related marker genes. The experiments described in this report follow
the latter approach, using the human Ad5 ElA gene as a candidate suppressor gene.
While the human Ad5 ElA gene is best known for its transforming properties and related
broad influences on gene regulation (for review, see ref. 11), the introduction of Ad5 ElA into
highly malignant rodent or human cells induced a reduction of the metastatic potential (re
viewed in ref. 12). The reduced metastatic activity in ElA transfected cells correlated with
reduced levels
e.
/ ■* and collagenase type I and type
I
Correspondence. Fax: +31-24 3540525. E-mail: [email protected].
Abbreviations used are: Ad5, adenovirus type 5; IL-6, interleukin-6; PAI-1, plasminogen activator inhibitor type
1; PAI-2, plasminogen activator inhibitor type 2; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulphate; tPA, tissue type plasminogen activator; Tgase2, tissue type transglutaminase; uPA, urokinase
type plasminogen activator.
808
0006-291X/96 $18.00
1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
i
mmà lim é
, No. 3,
BIOCHEMICAL
IV (14), or down-regulation of the proto-oncogene neu (15,16)
of the normal human c-erbB-2/neu proto-oncogene is a
cancers. Renewed ectopic overexpression of neu in
re-induced the
related,
EIA activity also stimulated nm23
tumor cells (18).
in
MATERIAL AND METHODS
i *
m
cell line was
on 12
cals Inc., Aurora
were incubated with the M73 anti-El A monoclonal antibody (24). Slides
incubated for 60 min
patts, Glostrup, Denmark). For actin staining, cells were
washing with PBS (3 X 10 min), slides were mounted in
RNA isolation and northern blot analysis. Total RNA was is
were
•y, UK).
m
4
AL
im
m
1
ml of hybridization mixture. To confirm that
hybridized to an
i
c -mv<
and BLM
expression in
1F6 and 530 (J. J. M.
Dr. R.
, mouse /3-actin, rat
fetal calf serum
*
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(BLM.mock)
018
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of
were grown in sufficient amounts to allow RNA isolation
cells). As expected, El A mRNA expression was only detectable in the three positive transfee
tants, BLM.E1-1
visible (Fig. 2A). In addition, we examined the expression of a panel of positive progression
marker genes, including calcyclin (4), thymosin /31Q (5), TGase2 (28), PAI-1 (30), PAI-2 (31),
Vol. 225, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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FIG. 2. Northern blot analysis of human melanoma cell lines, and E l A or E1A+E1B transfectants. Ten yug of total
RNA was loaded in each lane. Lane 1, 1F6; lane 2, MV3; lane 3, BLM; lane 4, BLM.mock; lane 5, BLM.neo; lane
6, BLMJE1-1; lane 7; BLM.E1-2; lane 8, BLM.E1A. (A) Expression of E1A, PA M , PAI-2, uPA, tPA, TGa-se2. (B)
Expression of memA, memB, thymosin /?10, calcyclin and c-myc. (C) Expression of p53, nma, nmb and ME49\. (D)
Expression of hsp70, ft actin and vimentin. Lambda Hindlll was used as a molecular marker; a 18S ribosomal probe
was used for control hybridization.
812
Vol. 225, No. 3,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I
Human Melanoma
and E l A or E l A + E1B
Ml
Cell line
IL-6
530
1F6
MV3
BLM
BLM.mock
BLM.neo
BLM.EI-1
BLM.EI-2
BLM.ElArev
<0.005
<0.004
13
9.0
14
10
<0.003
<0.006
8.4
mm
Cell lines were cultured for 48
was de. IL-6 values are given as
pg active protein per ml per 106 cells.
tPA
uPA
(two novel genes, associated with highly metastatic
M. van Groningen, unpublished results), vimentin and c-myc (29)
(note that c-myc showed no differential mRNA expression in our panel of human melanoma
The expression profile of BLM.E1-1, BLM.E1-2 and BLM.E1A showed a specific
switch off for most of these genes, while thymosin £10 and vimentin were downregulated to
non-metastatic cell line 1F6. The expression of some other genes was hardly
or not
actin, p53, nma and nmb (two novel genes, not expressed in our
and the reverse progression marker ME491 (33). In accor
% *
was greatly increased in El A expressing cells
In
the expression of positive progression marker genes is
. Gene activity in the non-metastatic cell
The secretion of IL-6 into the growth medium by the reference cell lines
transfectants was assayed by hybridoma growth stimulation (Table 1). The nonnot produce IL-6, whereas the highly metastatic cell
amounts of IL-6 into the growth medium. IL-6
C
md BLM.E l-2 was down-regulated to the level of the non-metastatic
to parental BLM, BLM.neo and BLM.mock. IL-6
530
was at the level of the parental BLM cell line.
two neo transfectants BLM.neo7
BLM.ElArev, and the El A expressing BLM.E1 and
9
formed large tumors within 6 weeks.
-1 and BLM.E1observations indicate that tumor growth of the
DISCUSSION
The presented set of experiments was designed to study molecular mechanisms controlling
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 225, No. 3, 1996
30
25
20
00
£
o
15
+
BLM
A
neo 7
O
nao 12
+
BLM.E1 Arev
0
fEz
BLM.E1 -1
3
O
>
10
O
BLM.E1 -2
5
0
0
10
30
20
40
50
days
FIG. 3. Growth curves of the tumors from parental BLM, BLM.neo 7, BLM.neo 12, BLM.ElArcv, BLM.E1-1 and
BLM.E1-2 cell lines in nude mice. Approximately 3 X 106 cells were inoculated s.c. into nude mice. Tumor volumes
were measured weekly and mice were allowed to sit for three months unless the tumor size interfered with the animal’
health. Values are given as a mean of all animals (5 animals per cell line) tested.
the highly metastatic human melanoma cell line BLM by stable transfection, we have observed
the apparently collective and coordinate suppression of a panel of genes whose expression is
normally positively related with the malignant BLM phenotype. While some of these positive
progression markers have a proven functional relation with metastasis, the functions of some
others, including two novel genes, are unknown in regard to neoplastic progression. The
apparently coordinate type of intervention is remarkable and novel, and provides evidence for
the existence of a common pathway of neoplastic progression in melanoma cells.
Significant differences existed in the levels of ElA expression in the three transfectants,
BLM.E1-1 displaying the highest levels of both mRNA and protein. Despite these variations
all three El A expressing transfectants were equally capable of suppressing positive progression
marker gene expression and changing cell morphology. As additional confirmation for the
presence of ElA activity we observed the well described stimulation of hsplO expression (35)
in all three transfectants, BLM .EM , BLM.E1-2 and BLM.E1A. The complete suppression of
several genes (e.g. u-PA, PAI-1) in all transfected cell clones is a strong argument in favor
of homogeneity of the cultures analyzed. The presence of the ElA gene had no effect on p53
transcript levels, but it is known that ElA stabilizes the p53 tumor suppressor protein and
promotes apoptosis (36). Continuous culturing may, therefore, select for cells that have lost
ElA expression. Accompanying the loss of ElA expression, BLM.ElArev had reverted to the
parental phenotype, further confirming that ElA activity is responsible for the observed pheno
typic changes. The Ad5 E1B gene products inhibit ElA associated apoptosis (37). Therefore,
transfectants and, indeed, ElA expression is apparently stable in BLM.E1-1 and BLM.E1-2
cell lines.
In addition to the differences in the gene expression profiles, the ElA transfectants display
changes in cell morphology and cytoskeletal organization. This phenomenon is not normally
related with melanoma progression (compare ref. 38). Apparently, ElA proteins do not only
cause suppression of late progression marker genes but have broader effects. This is not
surprising in view of the number and nature of the cellular factors that are known to associate
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with El A (11,39). El A induced phenotypic alterations were more often associated with a
cytoskeletal reorganization, the resultant patterns being cell-type-dependent (38,40).
Our data provide new evidence in support of a tumor suppressor function for E lA , as
suggested recently by several other groups (12,40-43). Reduced oncogenicity would be a likely
and logical consequence of the observed suppression of positive progression marker genes.
Subcutaneous inoculation of the transfected melanoma cells into nude mice indeed showed
that the presence of ElA significantly reduced tumor growth. The properties of the reverted
cell line BLM.E1 Arev corresponded also in this test with those of parental BLM.
to this subject no unifying concept has emerged yet. The ample data are listed in recent reviews
(11,39). In general, it is concluded that the mechanism does not seem to be mediated by one
specific factor or interaction. Furthermore, it is pointed out that the qualitative effect (suppression or promotion) is
factors (RB, p i07, pl30, p300). It is known that E lA inhibits cAMP-dependent activation of
the IL-6 promoter due to interac tions with and inhibition of CBP (44). The mechanisms of
the effects described in this report have to be elucidated in further studies.
In summary, our results add new evidence to the earlier observed suppression/reversion of
neoplastic progression by ElA (15,17,40,42,43,45). More importantly, while the apparent
inhibition of the oncogenic potential of several human tumor cell lines was ascribed to the
mere suppression of individual genes essential for advanced progression, our data show that
ElA interference is more comprehensive and concerns the specific suppression of all tested
panel of
genes whose expression is positively correlated with neoplastic progression
human melanoma cell lines of increasing metastatic potential. We therefore conclude that EIA
fulfills the role of a “ master gene” , i.e. a gene responsible for the transition of one phenotype
to another. The coordinate intervention realized by a viral gene suggests that neoplastic progres
sion proceeds along a common pathway(s), which may be equally well controlled by (an) as
yet unidentified cellular factor(s).
ACKNOWLEDGMENTS
The authors thank Dr. A. Zantema (Leiden, The Netherlands) for discussions and for providing the
pAd5Xho and pAdSPst and the M73 monoclonal antibody.
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