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Tumor Biology |
Department of Oncology/Pathology, Division of Cellular and Molecular Tumor Pathology, Karolinska Hospital, SE-171 76 Stockholm, Sweden [L. G., A. G., B. B., Y. X., G. N., A. D., J. W., A. B., O. L.]; Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden [J. L.]; and Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 77 Stockholm, Sweden [K. G. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IGF-IR has been shown to be crucial for tumor transformation and
maintenance of tumorigenicity, and it promotes cell growth and prevents
apoptosis (6, 7, 8)
. IGF-IR is composed of two
extracellular 
-subunits, which are involved in ligand binding, and
two transmembrane ß-subunits containing tyrosine kinase domains
involved in signal transduction (9, 10, 11)
. Recently, it was
shown that wt p53 repressed the transcriptional activity of the IGF-IR
gene, whereas mutant p53 had the opposite effect (12)
.
These studies were performed on p53-negative cells transfected with wt
or mutant p53 cDNA (12)
. The aim of present study was to
elucidate whether p53 could have any functional impact on tumor cell
growth through interaction with IGF-IR. We were particularly interested
in investigating whether overexpression of IGF-IR in malignant cells
could be dependent on expression of mutant p53.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IR-3),
p21Waf1, and Bcl-2 were purchased from Oncogene
Science. A polyclonal IGF-IR antibody (N-20), mouse monoclonal
antibodies against human p53 (DO1), a mouse monoclonal antibody against
Mdm-2 (including the p53-Mdm-2 complex), a monoclonal antibody against
phosphotyrosine (PY99), and an antibody against actin (H-196) were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A pan-CD44 (IM-7)
monoclonal antibody was from the American Type Culture Collection
(Manassas, VA). The proteasome inhibitor lactacystin was from
Calbiochem (Darmstadt, Germany). Unless otherwise stated, all other
reagents were from Sigma (St. Louis, MO).
Cell Lines.
The human melanoma cell lines SK-MEL-5 and SK-MEL-28, ES cell line
RD-ES, and the human p53-negative cell lines Saos-2 and HL-60 were
obtained from American Type Culture Collection. BL41-tsp53-2 is an
EBV-negative Burkitt lymphoma cell line carrying mutant p53 (codon 248)
transfected with ts p53 mutant (p53-Val135) with
mutant conformation at 37°C and wt conformation at 32°C
(13, 14, 15)
. The HDFs (GM08333) were obtained from the
Coriell Institute of Medical Research. BE cells, which were established
from a lymph node metastasis specimen from a patient with advanced
malignant melanoma (16)
, were kindly provided by Prof.
Rolf Kiessling (Karolinska Hospital, Stockholm, Sweden). SK-MEL-5,
SK-MEL-28, BE and GM08333 cells were cultured in MEM supplemented with
10% FCS, HL-60 cells were cultured in Iscove’s modified Eagle’s
medium supplemented with 15% FCS, Saos-2 cells were cultured in
McCoy’s 5a medium supplemented with 20% FCS, RD-ES cells were
cultured in RPMI 1640 supplemented with 10% FCS, and BL-41 cells were
cultured in RPMI 1640 with 10% FCS.
Immunoprecipitation.
The isolated cells were lysed as described elsewhere (17)
.
Protein G Plus-A/G-agarose (0.15 µl) and 1 µg of antibody were
added to 1 ml of protein material. After a 24-h incubation at 4°C on
a rocker platform, the immunoprecipitates were collected by
centrifugation in a microcentrifuge at 2500 rpm for 15 min. The
supernatant was discarded, and the pellet was washed. The material was
then dissolved in sample buffer for SDS-PAGE.
SDS-PAGE and Western Blotting.
Protein samples were dissolved in a sample buffer containing 0.0625
M Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, bromphenol
blue, and DTT. Samples corresponding to 50–100 µg of cell protein
were analyzed by SDS-PAGE with a 7.5% or 10% separation gel
essentially according to the protocol of Laemmli (18)
.
Molecular weight markers (Bio-Rad) were run simultaneously. After
SDS-PAGE, the proteins were transferred overnight to nitrocellulose
membranes (Hybond; Amersham) and then blocked for 1 h at room
temperature in a solution of 5% (w/v) skimmed milk powder and 0.02%
(w/v) Tween 20 in PBS (pH 7.5). Incubation with the appropriate primary
antibody was performed for 1 h at room temperature. This was
followed by washes with PBS and incubation with a biotinylated
secondary antibody (Amersham) for 1 h. After incubation with
streptavidin-labeled horseradish peroxidase, detection was
made (Hyperfilm-ECL; Amersham). The films were scanned by Fluor-S
(Bio-Rad).
Quantification of IGF-IR and p53 mRNA Levels.
Total RNA was isolated from the cells using RNazol according to the
manufacturer’s protocol (Qiagen, Hilden, Germany). Total RNA (500 ng)
was reverse transcribed into cDNA. Five µl of the cDNA reaction were
used to amplify IGF-IR (forward primer, 5'-GCCCGAAGGTCTGTGAGGAAGAA-3',
position 1028; reverse primer, 5'-GGTACCGGTGCCAGGTTATGA-3', position
1559), ß-actin (forward primer, 5'-CACGGAGTACTTGCGCTCAGGAGG-3';
reverse primer, 5'-CACGGAGTACTTGCGCTCAGGAGG-3'), and p53 (forward
primer, 5'-CCGAGTGGAAGGAAATTTGCGTGTGGAGTA-3', position 799; reverse
primer, 5'-CAAGGCCTCATTCAGCTCTCGGAACATCTC-3', position 1299).
Competitive PCR for quantification of IGF-IR and p53 transcripts was
performed as described elsewhere (19)
.
Antisense Experiments.
AS-ODNs (5'-CCCTGCTCCCCCCTGGCTCC-3') and S-ODNs
(5'-GGAGCCAGGGGGGAGCAGGG-3') to p53 were purchased from Pharmacia
Biotech. AS-ODN is complementary to position 1071–1090 of exon 10 of
the p53 mRNA (20
, 21) . Lipofectin (Life Technologies,
Inc.) was used to deliver AS-ODNs to cultured cells. Because AS-ODN
induces RNase H cleavage and further degradation of target mRNA, we
tested the specificity of the p53 AS-ODN using semiquantitative RT-PCR.
RNA was isolated from SK-MEL-5 and SK-MEL-28 cells that had been
treated with p53AS-ODN for 24 h. As shown in Fig. 1
, the p53 transcript was dramatically decreased after treatment with
AS-ODN. In contrast, no decrease was observed in the Lipofectin control
or after treatment with AS-ODN plus S-ODN.
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Isolation of Plasma Membranes.
Preparation of plasma membranes was performed essentially as described
elsewhere (23)
. In brief, cells were harvested and
homogenized. After a 10-min centrifugation at 600 x
g (4°C), the pellet (containing unbroken cells, nuclei,
and cytoskeleton) was discarded. The supernatant was then centrifuged
at 17,300 x g for 30 min. The resulting
pellet contains plasma membranes (23)
.
Assay of Cell Growth and Survival.
For assay of DNA synthesis, cells cultured in 35-mm dishes were labeled
with [3
H]thymidine (1 µCi/ml, 5 Ci/mmol)
during the last 4 h of the experiments. The acid-precipitable
material was then taken for scintillation counting as described
elsewhere (17)
. Proliferation of the cell lines, with the
exception of HDFs and BL-41tsp53-2, was measured by determining the
number of cells attached to the plastic surface of duplicate 35-mm
dishes. This was performed by microscopic counting of cells in several
ink-marked areas on the dish bottom. By repeating the countings after
specified time intervals, changes in the number of attached cells could
be followed (24)
. In HDFs, cell growth was assayed by
determining changes in cellular protein content, and in BL-41tsp53-2
cells, cell growth was assayed by counting cells in a Burker chamber.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit of IGF-IR, we investigated the expression of
IGF-IR at the cell surface by Western blotting, as described elsewhere
(17)
. A strong IGF-IR signal was seen in cells cultured at
37°C but was deleted at 32°C (Fig. 2A)
. As a control, we investigated IGF-IR expression in
parental BL-41 cells. As shown in the right panel of Fig. 2A
, there were no differences in the IGF-IR signals between
the two culture conditions in the parental cell line. Thus,
introduction of wt p53 in cells normally expressing mutant p53 leads to
IGF-IR down-regulation. In line with the findings of Werner et
al. (12)
, this indicates that IGF-IR is negatively
regulated by wt p53. However, a closer investigation of the role of
mutant p53 is justified. In Fig. 2B
, it is demonstrated that
p53 AS-ODN substantially decreased IGF-IR expression in BL-41tsp53-2
cells cultured at 37°C. IGF-IR expression was very low in
BL-41tsp53-2 cells cultured at 32°C and remained low in the presence
of AS-ODN (Fig. 2B)
. Taken together, these data suggest that
expression of functionally aberrant p53 is important for IGF-IR
expression in this cell line.
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. Overexpression of p53 is generally due to point
mutations. Isolated DNA from BE and SK-MEL-5 cells was sequenced for
analysis of exons 2–10 of TP53. Only one point mutation was found, and
this was localized in exon 7 of TP53 (codon 248,
CGG-TGG, Arg-Trp) in the BE cells (Fig. 3B)
. Interestingly, at the position of this point mutation,
there is only one signal detected, and this is for T (Fig. 3B)
. This means either that both alleles have the same point
mutation or that one allele is missing. In turn, this means that only a
single species of p53 is expressed in BE, i.e., codon
248-mutant p53. We did not find any point mutations in exons 2–10 of
SK-MEL-5 (data not shown). The same result was obtained by O’Connor
et al. (25)
, who performed a complete sequence
analysis of p53 in a large number of cell lines, including SK-MEL-5.
This suggests that other mechanisms are involved in p53 overexpression
in this cell line. The expression of p21Waf1 in
SK-MEL-5, as compared with HDFs and BL-41tsp53-2 cells (32°C), was
investigated. As expected, the BL-41tsp53-2 cells expressed
p21Waf1 on induction of wt p53 (Fig. 3C)
. In contrast, there was no detectable
p21Waf1 in HDFs and SK-MEL-5. Immunofluorescence
analysis demonstrated similar results (data not shown). The reason why
HDFs did not express p21Waf1 is most likely due
to the low p53 expression in these cells under normal growth conditions
(compare with Fig. 3A
). The absence of
p21Waf1 expression in SK-MEL-5 suggests that the
suppressor function of p53 is lost in these cells. A possible mechanism
involved in inactivation of wt p53 could be association with Mdm-2. In
Fig. 3D
, the expression of Mdm-2 in SK-MEL-5 is compared
with Mdm-2 expression in ES cells, the p53-negative osteosarcoma cell
line Saos-2, and HDFs. Whereas Mdm-2 expression was not detectable in
HDFs and ES cells and was weak in Saos-2 cells, this protein was
strongly expressed in SK-MEL-5 cells. We also investigated whether p53
was complex-bound to Mdm-2 in SK-MEL-5. This was assayed by
immunoprecipitation using an antibody to Mdm-2, followed by Western
blot analysis of p53. As shown in Fig. 3E
, there is a strong
p53 signal in SK-MEL-5 cells. These data suggest that inactivation of
p53 in SK-MEL-5 can be due to association with Mdm-2.
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As a control experiment, we first analyzed the effect of AS-ODN on
IGF-IR expression of the p53-negative cell line HL-60. We confirm that
AS-ODN had no effect on IGF-IR in these cells (Fig. 4
, top panel). In the middle panels of Fig. 4
, we
show that a 24-h treatment with AS-ODN down-regulated both p53 and
IGF-IR in BE and SK-MEL-5 cells. This effect was prevented on
coincubation with p53 S-ODN. Lipofectin per se did not
affect p53 or IGF-IR expression. In the bottom panels of
Fig. 4
, we show that the expression of three other proteins (Bcl-2,
CD44, and ß-actin) functioning as loading controls was not altered
after treatment with AS-ODN. Our results suggest that inhibition of the
expression of aberrant p53 leads to down-regulation of IGF-IR. AS-ODN
consistently decreased the level of IGF-IR transcripts (Fig. 5)
, suggesting that p53 may regulate IGF-IR at a transcriptional level.
However, this decrease was limited to 50–60%. This could be
explained simply by insufficient accuracy of the semiquantitative
RT-PCR method used. Alternatively, the observed 50–60% decrease in
transcripts may be sufficient to block the expression of the IGF-IR
protein. Another explanation is that the block of p53 may also inhibit
IGF-IR expression at posttranscriptional levels.
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strongly suggest that inhibition of p53
expression drastically reduces the number of IGF-IRs in BE and SK-MEL-5
cells. A Scatchard plot analysis would be helpful to prove this, but
binding analysis could be disturbed by p53-mediated changes of
expression of IGF-binding proteins. p53 has in fact been demonstrated
to regulate IGF-binding protein 3 (26)
. Instead, we
decided to investigate the effect of p53 inhibition on IGF-IR activity
by measuring the level of tyrosine phosphorylation of the ß-subunit.
This experiment was carried out on SK-MEL-5 and SK-MEL-28 cells.
SK-MEL-28 has a point mutation in codon 145 of p53 (27)
.
This cell line also overexpressed p53 and responded by down-regulation
of IGF-IR to p53 AS-ODN (data not shown). As shown in Fig. 6
, antisense treatment almost deleted IGF-I-mediated tyrosine
phosphorylation in both cell lines. This suggests that p53-mediated
changes of IGF-IR expression have functional impact.
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, the proteasome inhibitor lactacystin, which inhibits all three types
of proteasomes (30)
, moderately increased the expression
of wt p53 in SK-MEL-5 (by almost 40% as assayed by densitometry) but
did not increase the expression of mutant p53 in SK-MEL-28. In
SK-MEL-5, this was correlated with a drastic decrease in tyrosine
phosphorylation of IGF-IR (Fig. 7
, top panel). In contrast,
the level of phosphorylated IGF-IR in SK-Mel-28 was not significantly
changed.
|
. In contrast, the growth of BE, SK-MEL-5, and BL-41tsp53-2
[37°C (Fig. 8
, B—D)] treated with AS-ODN for
24–72 h was blocked. Nevertheless, after 48 h, a massive cell
loss was seen. Closer microscopic analysis revealed that the detached
cells had undergone cell death (data not shown). The decrease in the
effect of AS-ODN on BL-41tsp53-2 after a 72-h treatment is probably due
to degradation of AS-ODN. The effect on DNA synthesis, as assayed by
incorporation of [3
H]thymidine, was also
investigated. A 24-h exposure to p53 AS-ODN drastically decreased the
rate of DNA synthesis in BE, SK-MEL-5, and SK-MEL-28 cells (Fig. 8
,
E, F, and H) but not in p53-negative HL-60 cells
(Fig. 8G)
.
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IR-3 (which inhibits the
binding of IGF-I to IGF-IR; Ref. 31
) and lovastatin, on
the growth of BE and SK-MEL-5 cells. It was demonstrated previously
that lovastatin blocks cell surface expression of IGF-IR and that this
mechanism strongly contributes to lovastatin-induced growth arrest in
melanoma cells (17
, 24
, 32)
. As demonstrated in Fig. 9
, all three agents caused inhibition of DNA synthesis, growth arrest,
and cell death.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our present study, we investigated the functional impact of p53 on IGF-IR and IGF-IR-related events (cell growth and survival) in human malignant cells. To evaluate the effect on IGF-IR, we measured the amounts of IGF-IR at the cell surface in three different experimental systems. These involved: (a) a cell line with mutant p53 transfected with ts p53; (b) two malignant melanoma cell lines overexpressing p53 with point mutations, one of which only expressed mutant p53; and (c) a malignant melanoma cell line expressing functionally aberrant wt p53. We could demonstrate that induction of functionally normal wt p53 resulted in down-regulation of IGF-IR at the cell surface. This is in line with the observation of Werner et al. (12) showing that wt p53 represses the transcription of the IGF-IR gene. Their study was performed on p53-negative cells transfected with wt or mutant p53 cDNA, and the transcriptional effect was assayed using a construct carrying a luciferase reporter gene driven by the IGF-IR promoter (12) .
To elucidate whether expression of functionally aberrant p53 could influence the expression of IGF-IR, the three malignant melanoma cell lines and BL-41 tsp53-2 cells, all of which overexpressed IGF-IR at the cell surface, were investigated. In SK-MEL-5, which did not exhibit any point mutations, Mdm-2 was overexpressed and coimmunoprecipitated with p53. This indicates that p53 is inactivated due to complex binding to Mdm-2. Antisense oligonucleotides against p53 resulted in a drastic down-regulation of IGF-IR expression at the cell surface of all four cell lines. This was accompanied by reduced tyrosine phosphorylation of IGF-IR, a drastic decrease in DNA and cell replication, and, ultimately, by massive cell death. Similar responses were achieved by blocking the IGF-I pathway through other means. Our results suggest that IGF-IR expression can be a gain of function mechanism of aberrant p53 in malignant cells. This mechanism might help to protect malignant cells from apoptosis. A study by Prisco et al. (34) showing that 32D murine hemopoietic cells with constitutively expressed IGF-IR were resistant to the apoptotic effect of wt p53 after interleukin-3 withdrawal supports this notion.
It has been shown that mutant p53, in contrast to wt p53, stimulates the transcriptional activity of the IGF-IR gene (12) . However, according to our present study, p53 does not need to be mutated to increase IGF-IR expression. It seems that expression of functionally aberrant p53, i.e., p53 lacking its suppressor function (through point mutations or interaction with Mdm-2) may be of importance. This notion is supported by our finding that inhibition of proteasomes, which blocks Mdm-2 targeting of wt p53 (28 , 29) , decreased the IGF-IR activity in SK-MEL-5 cells.
Previous studies from our laboratory and other laboratories have shown that IGF-IR is crucial for the growth and survival of melanoma cells (17 , 24 , 32 , 35, 36, 37, 38) . The incidence of p53 mutations in melanoma is very low, whereas p53 overexpression is common (39, 40, 41, 42) . A possible mechanism underlying loss of suppressor function of p53 in melanoma might be interaction between p53 and Mdm-2. Based on our present study, this may cause up-regulation of IGF-IR, which in turn may contribute to the malignant phenotype of melanoma.
We believe that the mechanisms mediating the regulatory effect of p53 on IGF-IR are complex. Therefore, further exploration of the involved molecular mechanisms for p53-dependent IGF-IR expression in malignant cells is needed. Another possibility that remains to be investigated is whether aberrant p53 interferes with regulatory steps at posttranscriptional levels.
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FOOTNOTES |
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1 Supported by grants from the Swedish Cancer
Society, the Cancer Society in Stockholm, the Swedish Radiation
Protection Institute, and the Karolinska Institute. 
2 To whom requests for reprints should be
addressed, at Department of Oncology/Pathology, Division of Cellular
and Molecular Tumor Pathology, CCK, R8:04, Karolinska Hospital, SE-171
76 Stocholm, Sweden. Fax: 46-8-7588397; E-mail: olle.larsson{at}onkpat.ki.se
3 The abbreviations used are: wt, wild-type; IGF,
insulin-like growth factor; IGF-IR, insulin-like growth factor I
receptor; ts, temperature-sensitive; AS-ODN, antisense oligonucleotide;
S-ODN, sense oligonucleotide; HDF, human diploid fibroblast; RT-PCR,
reverse transcription-PCR; ES, Ewing’s sarcoma.
Received 1/ 3/00. Accepted 7/14/00.
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  N. P. Agaram, M. P. Laquaglia, B. Ustun, T. Guo, G. C. Wong, N. D. Socci, R. G. Maki, R. P. DeMatteo, P. Besmer, and C. R. Antonescu Molecular Characterization of Pediatric Gastrointestinal Stromal Tumors Clin. Cancer Res., May 15, 2008; 14(10): 3204 - 3215. [Abstract] [Full Text] [PDF]
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 L. Girnita, S. K. Shenoy, B. Sehat, R. Vasilcanu, D. Vasilcanu, A. Girnita, R. J. Lefkowitz, and O. Larsson beta-Arrestin and Mdm2 Mediate IGF-1 Receptor-stimulated ERK Activation and Cell Cycle Progression J. Biol. Chem., April 13, 2007; 282(15): 11329 - 11338. [Abstract] [Full Text] [PDF]
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A. Girnita, C. All-Ericsson, M. A. Economou, K. Astrom, M. Axelson, S. Seregard, O. Larsson, and L. Girnita The Insulin-Like Growth Factor-I Receptor Inhibitor Picropodophyllin Causes Tumor Regression and Attenuates Mechanisms Involved in Invasion of Uveal Melanoma Cells Clin. Cancer Res., February 15, 2006; 12(4): 1383 - 1391. [Abstract] [Full Text] [PDF]
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L. Girnita, S. K. Shenoy, B. Sehat, R. Vasilcanu, A. Girnita, R. J. Lefkowitz, and O. Larsson {beta}-Arrestin Is Crucial for Ubiquitination and Down-regulation of the Insulin-like Growth Factor-1 Receptor by Acting as Adaptor for the MDM2 E3 Ligase J. Biol. Chem., July 1, 2005; 280(26): 24412 - 24419. [Abstract] [Full Text] [PDF]
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