ORIGINAL ARTICLE
Journal of
The PVT-1 Oncogene Is a
Myc Protein Target That
Is Overexpressed in
Transformed Cells
Cellular
Physiology
LETIZIA CARRAMUSA,1 FLAVIA CONTINO,1 ARIANNA FERRO,1 LUIGI MINAFRA,1
GIOVANNI PERCONTI,1,2 AGATA GIALLONGO,3 AND SALVATORE FEO1,3*
1
Dipartimento di Oncologia Sperimentale e Applicazioni Cliniche, Università di Palermo, Palermo, Italy
2
Dipartimento Oncologico La Maddalena, Palermo, Italy
3
Istituto di Biomedicina e Immunologia Molecolare del Consiglio Nazionale delle Ricerche, Palermo, Italy
The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations
occurring in variant Burkitt’s lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription
over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc
expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low
number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed
cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to
identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites
for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in
proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins.
J. Cell. Physiol. 213: 511–518, 2007. ß 2007 Wiley-Liss, Inc.
The mis-1/pvt-1 locus was originally identified as a common
region of proviral integration in retrovirally induced lymphoma
of mice and rats (Villeneuve et al., 1986) and a breakpoint site in
variant t(6;15) translocations in mouse plasmacytomas
(Siwarski et al., 1997). The human mis-1/pvt-1 homologue,
termed PVT-1, is located on chromosome 8 about 55 kb distal
to the c-Myc gene (Shtivelman et al., 1989; Feo et al., 1994) and it
is frequently involved in the translocations occurring in variant
Burkitt’s lymphomas (Graham and Adams, 1986; Boehm and
Rabbitts, 1989). Furthermore, co-amplification of c-Myc and
PVT-1 has been found in a variety of human and animal tumors
(Shtivelman and Bishop, 1989; Bakkus et al., 1990; Huppi et al.,
1993; Storlazzi et al., 2004).
According to these data it has been proposed a functional
relationship between c-Myc and PVT-1 in which putative PVT-1
protein(s) may activate c-Myc gene transcription or,
alternatively, PVT-1 gene may interfere with normal
transcriptional regulation by long-range cis-acting effects
(Huppi et al., 1990; Shtivelman and Bishop, 1990).
Several PVT-1 cDNAs have been isolated from human
placenta and tumors with c-Myc/PVT-1 amplification
(Shtivelman and Bishop, 1990), Burkitt’s lymphoma carrying the
t(2;8) translocation (Shtivelman et al., 1989) and mouse spleen
(Huppi et al., 1990). Even so, the nature of PVT-1 transcripts in
normal cells remains unclear and the effect of PVT-1
deregulation on c-Myc expression has not been clarified.
Comparative sequence analysis of the genomic sequence versus
more than 185 cDNA clones (ESTs) indicates that the human
PVT-1 gene contains more than 48 exons and may produce, by
alternative splicing, at least 27 different transcripts putatively
encoding 26 different protein isoforms (AceWiew database:
LOC441378). Interestingly among the several ESTs that have
been assigned to the PVT-1 UniGene cluster (NCBI: UniGene
Cluster Hs.369836 Homo sapiens) a number of them may
potentially encode a protein showing high homology with
ß 2 0 0 7 W I L E Y - L I S S , I N C .
Annexin-A2 (ANXA2). This is a Ca(2þ)-binding protein that is
consistently up-regulated in virally transformed cell lines and in
human tumors. Recently it has been shown that ANXA2 binds
directly to both ribonucleotide homopolymers and human
c-myc RNA, playing a role in c-Myc expression control
(Filipenko et al., 2004). In addition, high level amplification and/
or overexpression of the PVT-1 gene have been significantly
associated with invasive breast cancer (Yao et al., 2006) and
reduced survival time in ovarian cancer patients (Gray et al.,
2006).
All these observations are indicative of a potential
involvement of PVT-1 in the maintenance of a transformed
phenotype. In this study we show that PVT-1 is a downstream
target of both c-Myc and N-Myc genes and we provide the first
evidence of PVT-1 transcriptional regulation by N-Myc in
neuroblastoma cells.
L. Carramusa and F. Contino contributed equally to this work.
Contract grant sponsor: Ministero dell’Università e della Ricerca
(MIUR) and POR Sicilia (misura 3.14-DiaMol).
Letizia Carramusa’s present address is Weizmann Institute of
Science, Department of Molecular Cell Biology, Rehovot, 76100,
Israel.
*Correspondence to: Salvatore Feo, Università di Palermo, Dip.
Oncologia Sperimentale e Applicazioni Cliniche, Via San Lorenzo
Colli, 312, 90146 Palermo, Italy. E-mail: [email protected]
Received 20 August 2006; Accepted 4 April 2007
DOI: 10.1002/jcp.21133
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Materials and Methods
Cell lines and culture conditions
U2OS, Raji, U-937, HL60, LAN-5, and SK-N-SH cell lines were
obtained from the American Type Culture Collection (Rockville,
MD). Raji, U-937 and HL60 cells were maintained in RPMI-1640
medium (Sigma, St. Louis, MO), containing 2 mM L-glutamine and
10% fetal calf serum (FCS) U2OS, LAN-5, and SK-N-SH, were
maintained in DMEM medium (Sigma) containing 2 mM L-glutamine,
0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and
10% FCS. Promyelocytic HL60 cells were differentiated towards
the granulocytic pathway by culturing for 3 days in the presence of
1.25% dimethyl sulfoxide (DMSO, Sigma). Leukemic U-937 cells
were differentiated into monocytes culturing for 2 days in medium
supplemented with 1.6 107 M 12-O-tetradecanoylphorbol
13-acetate (TPA, Sigma). Differentiation of LAN-5 and SK-N-SH
neuroblastoma cells was carried out by addition of 1 106 M
all-trans retinoic acid (RA, Sigma) to subconfluent cells. The culture
medium was changed every day and cells harvested at different time
intervals.
RNA dot blot analysis
The MTE-Array2 human multiple tissue expression array
(Clontech Laboratories, Mountain View, CA), was analyzed
according to the manufacturer’s instructions. The array filter was
hybridized under high stringency condition with the following
[a-32P]dCTP (Amersham Biosciences, Buckinghamshire, UK)
labeled probes: a 450-bp cDNA fragment corresponding to exon I
and II of the human PVT-1 gene (Shtivelman et al., 1989), a 1-Kb
cDNA fragment corresponding to exon II and III of the human cMyc gene (Feo et al., 1996). Specific mRNA amount of each tissue
or cell line in the array was normalized by hybridization with a
human Ubiquitin cDNA fragment, provided by the manufacturer as
a control. After hybridization and washing under high stringent
conditions, the blot was exposed to X-ray film. Autoradiographies
were scanned and the images were analyzed with the QuantityOne software (Bio-Rad, Hercules, CA).
Total RNA isolation, RT-PCR and quantitative real-time-PCR
Total RNA was extracted from 1 107 cells using the Trizol
reagent according to the manufacturer’s instructions (Invitrogen,
Carlsbad, CA). After quantification by spectrophotometry, RNA
(500 ng) was reverse-transcribed into cDNA by using Superscript II
reverse transcriptase (Invitrogen) as described previously
(D’Agostino et al., 2003). Amplification reactions were performed
in a 50 ml reaction volume containing about 50 ng total RNA
equivalents (3 ml of the cDNA mixture), 160 mM dNTPs, 1.0 mCi of
[a32P]dCTP, 10 pmol of the appropriate oligonucleotide primers,
1.1 mM MgCl2, 0.01% gelatin and 2.5 units of RedTaq polymerase
(Sigma). The oligonucleotide primers and cycle number for PVT-1,
c-Myc and N-Myc were as follows: PVT-1, forward—CAT GGT
TCC ACC AGC GTT ATT C, reverse—TCC TTG CGG AAA
GGA TGT TGG, 20 cycles; c-Myc, forward—CAG CAG AGC
GAG CTG CAG CC, reverse—CTG TCT TTG CGC GCA GCC
TG, 20 cycles; N-Myc: forward—CAG CAG AGC GAG CTG
CAG CC, reverse—CTG TCT TTG CGC GCA GCC TG, 18
cycles. The thermal cycle profile employed a 10 min denaturing step
at 948C followed by the number of cycles indicated above (928C for
30 sec, 628C for 30 sec, 728C for 30 sec) and by an extension step of
10 min at 728C. To correct for the experimental variations
between samples, oligonucleotide primers for glyceraldeidephosphate-dehydrogenase (GAPDH) were included in each PCR
reaction: forward—TGA CAT CAA GAA GGT GGT GA,
reverse—TCC ACC ACC CTG TTG CTG TA. The amplimers
were separated on a 6% polyacrylamide gel in 1 TBE buffer
(45 mM TrisHCl, 45 mM boric acid, 1 mM EDTA). The amount of
[a-32P]dCTP incorporated into each amplimer was measured by
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
short exposure to X-Omat AR films (Kodak, Science Park, NH)
and analysis with the Quantity-One software (Bio-Rad).
The FluoCycle SYBR-Green hot-start Taq ready-mix kit for
quantitative real-time PCR was used following the manufacturer’s
instructions (UeroClone, IT). The primers and conditions for
real-time PCR were the same as described above for RT-PCR.
Real-time PCR was performed in a SmartCycler II system (Cepheid,
Sunnyvale, CA). All data shown were generated from three
independent experiments and are expressed as mean SD.
Plasmids construction and transfections
Fragments of the human PVT-1 promoter and 50 -flanking region
were inserted into the pGL3-Basic vector (Promega, Madison, WI),
upstream of the firefly luciferase gene, and named according to the
fragment length. In detail plasmid pGL-Pvt(1,764/þ132),
containing PVT-1 exon I, was created by insertion of a 1.9-Kb PstI/
BglII DNA fragment, isolated from the P1 genomic clone
ICRFP700.350 (Feo et al., 1994), into the PstI/BamHI sites of the
pGL3-basic vector. The complete sequence of the 1.9 kb PstI/
BamHI DNA fragment has been submitted to Genebank/EBI data
bank with accession number AY33608. Plasmids pGL-Pvt(1,266/
þ132), pGL-Pvt(615/þ132), pGL-Pvt(296/þ132) and pGLPvt(15/þ132) were created by digestion of the promoter
fragment contained in plasmid pGL-Pvt(1,764/þ132) with the
restriction enzymes PstI and AflII, KpnI, NotI, and SacII,
respectively. The generated fragments were gel purified, blunt
ended with T4 DNA polymerase (Roche, Basel, CH) and religated.
Plasmids pGL-Pvt(296/þ132)-E-box1-mut and –E-box2-mut
were obtained by PCR using two 35-bp double stranded
oligonucleotides containing sequence from the PVT-1 promoter
with a mutated E-box (wild type: 50 -CACGCG-30 , mutated:
50 -CCCGGG-30 ) and the QuickChange site-directed mutagenesis
kit (Stratagene), according to manufacturer’s instructions. The
plasmid 4xE/TK-Luc, containing the TK promoter and four E-box
consensus sequences has been described previously (Mori et al.,
1998). All constructs were sequenced to confirm sequence
deletions and mutations. LAN-5, U2OS and Rat1-MycER cells were
transfected with Lipofectamine 2000 reagent in OptiMem medium
(Invitrogen) as instructed by the manufacturer. In all cases, 1 mg
of the appropriate reporter construct was transfected in duplicate
along with 0.5 mg of the b-galactosidase expression vector pON-1
(Spaete and Mocarski, 1985) to correct for variability in
transfection efficiency. Raji cells (1 106) were transfected by
electroporation with 20 mg of the reporter construct and 5 mg
of pON-1 in 0.4 ml of OptiMem medium at 720 mF and 250 V in a
Bio-Rad electroporator. Cell extracts were prepared 48 h after
transfection and Luciferase activity was measured in duplicate for
all samples in a Turner 20/20 luminometer (Turner Designs, Inc.,
Sunnyvale, CA) using the Promega luciferase assay system. Betagalactosidase activity was assayed as described previously (Feo
et al., 1995). The ratio of luciferase activity to b-galactosidase
activity in each sample served as a measure of the normalized
luciferase activity. All data shown were generated from four to six
independent experiments, using at least two different plasmid
DNA preparations, and are expressed as mean SD.
Western blot analysis
Western blot analysis was performed as described previously (Feo
et al., 2000). Briefly, total cell lysates (30 mg) obtained from LAN-5
cells by extraction in RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM
NaCl, 1% NP-40, 0,5% DOC, 0.1% SDS) containing a protease
inhibitor mixture (Roche), or nuclear extracts from RAT1-MycER
cells (10 mg), prepared as described previously (Feo et al., 1995),
were resolved by electrophoresis on SDS–polyacrylamide gel and
electroblotted to a nitrocellulose membrane (Hybond-C,
Amersham Bioscience). The membrane was incubated with affinity
purified antibodies against c-Myc, N-Myc (Santa Cruz, Cat. nos. sc764 and sc-791, respectively) or anti-HSP70 (SIGMA), as a control
PVT-1 EXPRESSION IN NORMAL AND TRANSFORMED HUMAN CELLS
for loading, and then with horseradish peroxidase-linked
secondary antibodies (Amersham Bioscience). The
antigen-antibody complexes were visualized by enhanced
chemiluminescence (Pierce, Rockford, IL).
Chromatin immunoprecipitation (ChIP) assay
Molecular interaction between N-Myc and cis-element on the
PVT-1 promoter was investigated in vivo by using a ChIP assay kit
(Upstate Biotech, Billerica, MA). About 1 107 proliferating and
differentiated LAN-5 cells cultured on 10-mm dishes were treated
with 1% formaldehyde for 10 min at 378C to cross-link proteins to
chromatin. After rinsing with 125 mM glycine in PBS cells were
washed with cold PBS and lysed in SDS lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-HCl, pH 8.1). The lysate was sonicated to shear
DNA to a length between 200 and 600 bp. The sonicated
supernatant was diluted 10-fold with ChiP dilution buffer (0.01%
SDS, 1% Triton X-100, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) and
incubated, after a preclearing step with salmon sperm DNA/
proteinA-agarose (Upstate Biotechnology) either with anti-N-Myc
antibodies (Santa Cruz, Cat. no. sc-791), anti-acetylated H4
antibodies (Upstate Biotechnology, Cat. no. 06-866) or preimmune
serum overnight at 48C. To collect DNA-antibodies complexes a
salmon sperm DNA/proteinA-agarose slurry was added to the
mixture, incubated for 1 h at 48C with rotation and DNA/
proteinA-agarose complexes were recovered by centrifugation.
After extensive washing the pellet was dissolved in 0.25 ml
of elution buffer (0.2% SDS, 0.1 M NaHCO3), and the suspension
was spun to remove agarose. Supernatant was made 0.2 M with
NaCl and incubated a 658C for 4 h to reverse cross-linking. After
proteinase K treatment DNA was extracted with phenol/
chloroform and precipitated with ethanol. For PCR one-tenth of
the recovered DNA was amplified using specific primers directed
to region 220/35 of the PVT-1 promoter (forward: TCT CCG
GCT CAG TGC CCT GCG CT, reverse: CTG GCG GGT TGC
CCG TGA CGT); primers AP1107/AP1108 targeted to a region of
the ENO1 promoter containing an high affinity E-box (Fernandez
et al., 2003) as a positive control; primers directed to region þ113/
þ267 of PVT-1 exon I (forward: TCC TTG CGG AAA GGA TGT
TGG CGG, reverse: TGG AGG GCA GAT CTG GCC GTG), as a
negative control. To verify that an equivalent amount of chromatin
was used in the immunoprecipitations, DNA samples representing
0.1% of the total input chromatin was included in the PCR
reactions. The thermal cycling parameters were 958C for 10 min,
Fig. 1. Expression levels of PVT-1 and c-Myc in normal human tissues and cell lines. A human multiple tissue expression array (MTE-Array2,
Clontech) containing poly(AR) RNA from 76 human tissues and cell lines was sequentially hybridized with radiolabelled cDNA probes specific for
human PVT-1 (A), human c-Myc (B) and Ubiquitin (C), as described in Materials and Methods. Each probe was used in two separate experiment to
confirm results. See the manufacturer website for details about the mRNA samples and positions on the array (www.clontech.com/clontech/
archive/JAN99UPD/humanmte.shtml). D: Comparative analysis. Autoradiographies were scanned and quantitative analysis was performed. Dots
intensity was normalized with respect to the signals obtained by hybridization with the radiolabelled Ubiqitin cDNA probe. Relative PVT-1 and
c-Myc mRNA levels have been compared in tissues and cell lines which express detectable PVT-1 mRNA.
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
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followed by 30 cycles at 958C for 30 sec, 608C for 30 sec and 728C
for 30 sec. Amplification products were analyzed in 2% agarose gel
and DNA bands were documented using a gel documentation
system (AlphaImager, SAlpha Innotech, San Leandro, CA). To
quantitate immunoprecipitated chromatin real-time PCR was
performed with one-tenth of the recovered DNA per reaction and
100 nM of the primers described above diluted in a final volume of
25 ml in SYBR-Green hot-start Taq ready-mix (FluoCycle,
Euroclone, IT). Accumulation of fluorescent products was
monitored by using a SmartCycler system (Cepheid, CA). The
thermal cycling parameters were as described above with the
exception that 40 cycles were used. In these conditions each
primer set produced a single product, as determined by melt-curve
analysis and gel electrophoresis. Data are presented as percent of
total input DNA calculated has described by Frank et al. (2001).
Results and Discussion
PVT-1 and c-Myc expression in normal human tissues
and transformed cells
It has been shown that a probe representing the first exon of the
PVT-1 gene detects a heterogeneous pattern of polyadenylated
RNAs in a variety of human cell lines, (Shtivelman and Bishop,
1989). The most prominent transcript has a length of 4.8 kb and
the others have lengths ranging from 1 to 9 kb, none of these
mRNA has been isolated and completely sequenced to date.
This heterogeneity is due to the complex structure of the
PVT-1 gene which results in the generation of many alternative
spliced mRNAs. Therefore, to elucidate the putative functional
relationship between PVT-1 and c-Myc genes, we thought in the
first place to investigate the relative presence of transcripts in a
wide panel of normal and transformed cells. We analyzed the
expression of both genes by using a commercially available
‘‘Multiple Tissue Array’’ membrane containing normalized
amount of poly(A)þ mRNA from several normal tissues and
cancer cell lines, all of human origin. The filter was sequentially
hybridized with probes corresponding to the first exon of the
PVT-1 gene (Fig. 1A), the second and third exon of the c-Myc
gene (Fig. 1B), and at last with a probe for Ubiquitin (Fig. 1C),
commonly used as internal positive control (Blanquicett et al.,
2002). Signals quantification was performed and all the
collected data were normalized for loading with respect to the
Ubiquitin control. Relative PVT-1 and c-Myc mRNA levels have
been compared only for tissues which express PVT-1 and
results are shown in Figure 1D. PVT-1 mRNA was undetectable
in all the tissues of neuronal origin (Fig. 1A, 1, 2, and 3 from A to
H), whereas c-Myc was detected in cerebral cortex (Fig. 1B, 1B),
parietal lobe (Fig. 1B,D), cerebellum (Fig. 1B, 2B), corpus
callosum (Fig. 1B, 2C) and caudate nucleus (Fig. 1B, 2E). Among
the other tissues low to moderate PVT-1 expression was
observed with the exception of the adrenal gland where a
higher level of PVT-1 transcript was observed compared to the
c-Myc level (Fig. 1A and B, 9C). Interestingly, while c-Myc
mRNA was detectable in all the foetal tissues examined
(Fig. 1B, row 11) with the exception of the foetal brain
(Fig. 1B, 11A), PVT-1 mRNA was detected only in foetal kidney
(Fig. 1A, 11C) and foetal spleen (Fig. 1A, 11E). PVT-1 expression
was from two- to fourfold higher than the average in a variety of
Fig. 2. Analysis of PVT-1 and c-Myc expression in human cancer cell lines. Total cellular RNA was extracted, reverse transcribed and amplified
with gene specific primers as described in Materials and Methods. A: Semiquantitative RT-PCR showing expression levels of PVT-1 and c-Myc
mRNAs in hepatocarcinoma cells Hep-G2; breast cancer cells SkBr3 and BC-8701; lung carcinoma cells N-417; osteosarcoma cells U2OS;
neuroblastoma cells LAN-5; Burkitt’s lymphoma cells Manca, Daudi and Raji; erytroleukemia cells K562. B: Expression of PVT-1 and c-Myc in
proliferating and differentiated promyelocytic cells HL60 (3-day DMSO treatment) and monoblastic leukaemia cells U-937 (2-day TPA
treatment). C: Quantitative real-time PCR analyses of PVT-1 and c-Myc expression. Real-time PCR was performed using the same cDNAs and
primers asusedin thecase ofsemiquantitative RT-PCR(see Materials and Methods). All samples were normalized with respect to thedata obtained
by amplification with GAPDH specific primers. Data regarding U-937 and HL60 are referred to proliferating (U-937 and HL60, respectively)
and differentiated (U-937 diff. and HL60 diff., respectively) cells. Data were generated from three independent experiments, and the values are
expressed as mean W SD.
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
PVT-1 EXPRESSION IN NORMAL AND TRANSFORMED HUMAN CELLS
Fig. 3. Functional analysis of the PVT-1 promoter in various human cell lines. A: Graphic representation of PVT-1 exon-1 and 5(-flanking region.
Position of the restriction sites used to generate deletion constructs and putative consensus sequences for known transcription factors are
indicated. B: Functional analysis by transient transfection into U2OS (striped bar), Raji (dotted bar) and LAN-5 (squared bar) cells. Fragments of the
human PVT-1 promoter and 5(-flanking region were inserted into the pGL3-Basic vector (Promega), upstream of the firefly luciferase gene, and
named according to the fragment length (see Materials and Methods). A beta-galactosidase reporter plasmid was co-transfected to correct for
differences in transfection efficiencies. Luciferase activity is expressed as a percentage of the activity obtained in each cell line with the pGL3-control
plasmid, driven by the SV40 promoter and enhancer. Standard deviations were calculated out of three to four independent experiments.
transformed cells (Fig. 1A, row 10), such as HL60 (Fig. 1A, 10A),
HeLaS3 (Fig. 1A, 10B), K-562 (Fig. 1A, 10C) Raji (Fig. 1A, 10E),
SW480 (Fig. 1A, 10G) and A549 (Fig. 1A, 10H), which all
express c-Myc at high levels. As expected, no signals were
detected in all the negative controls (Fig. 1A, 1B, row 12).
Overall, these results revealed a substantial difference in the
expression patterns of PVT-1 and c-Myc genes in normal
tissues: c-Myc is expressed at a relatively high level in many
tissues whereas PVT-1 mRNA is less abundant, or barely
detectable. On the contrary in transformed cell lines
overexpressing c-Myc, elevated levels of PVT-1 transcripts are
also present.
We further investigated the expression of PVT-1 and c-Myc
genes in human cancer cell lines derived from different tumors
by semiquantitative RT-PCR analysis. In all the cell lines
examined, with the exception of the breast cancer BC-8701,
high levels of PVT-1 mRNA were detected (Fig. 2A). These
results indicate that the abundance of PVT-1 mRNA in
transformed cells well correlates with the relative levels of
c-Myc or N-Myc mRNA, as shown for LAN-5 cells which
overexpress N-Myc (see Fig. 5A), rather than with the presence
of structural alterations involving PVT-1 and/or c-Myc genes,
that is, the rearrangements peculiar to Burkitt’s lymphomas
(Manca, Raji and Daudi), or the amplification events
characterizing the breast cancer cells SKBr3 and the lung cancer
cells N-417 (Fig. 2A). Comparative analysis of c-Myc and PVT-1
mRNA levels was also performed in proliferating and
differentiated promyelocytic HL60 and monoblastic U-937
cells. The experiment results show a consistent correlation
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
between down-regulation of c-Myc expression and decrease of
PVT-1 transcripts (Fig. 2B). Then we performed quantitative
real-time PCR on all the isolated RNA samples described above
using gene-specific primers. The results are shown in
Figure 2C. The real-time PCR data corroborate the RT-PCR
results and all together these data support the hypothesis that
rather than a c-Myc activator the PVT-1 gene might be indeed a
target of Myc and its expression might depend on the relative
abundance of Myc proteins in the cells.
Deletion analysis of the human PVT-1 promoter in
various cell lines
As first approach towards the understanding of the PVT-1/Myc
interplay a functional analysis of the PVT-1 promoter was
performed. To identify DNA sequences important for basal and
regulated expression of PVT-1 in human cells, we constructed a
series of luciferase reporter plasmids containing serial
50 -deletions covering the promoter region. A 1.9 Kb PstI/BglII
DNA fragment, containing PVT-1 exon I and its 50 -flanking
region (Feo et al., 1994) was subcloned into the pGL3-basic
vector, deletion mutants were obtained by sequential digestion
and ligation as depicted in Figure 3A. The luciferase reporter
plasmids were introduced into the osteosarcoma cells U2OS,
the Burkitt’s lymphoma cells Raji and the neuroblastoma cells
LAN-5. In all three cell lines the activity of the longest PVT-1
genomic fragment, spanning from nt 1,764 to nt þ132 with
respect to the human gene transcription start site (Shtivelman
and Bishop, 1989), was 10–30% that of the pGL3 vector driven
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by the SV40 promoter/enhancer used as a control. Serial
50 -deletions from nt 1,764 to nt 1,266 and from nt 1,266
to nt 615 resulted in a significant increase of luciferase activity
in Raji and LAN-5 cells (40–60% and 80–135%, respectively),
but not in the U2OS cells, suggesting the presence of negative
regulatory elements acting in neuronal and lymphoid cells.
Deletion of sequences between nt 615 and nt 296 resulted
in a significant reduction of luciferase activity in Raji cells (20%
the activity of the control) and in a further increase of activity in
LAN-5 cells (220%). A further deletion of sequences between
nt 264 and nt 15 caused the loss of promoter activity in all
three cell lines.
These results suggest that the region between nt 615 and
nt 296 may play a relevant role in transcriptional regulation of
the PVT-1 promoter in B-cells. Our findings are consistent with
the presence in this region of consensus binding sites for several
well characterized lymphoid-specific regulatory factors like
GATA-3, Ikarus-1, NFk-B and Ets (Rothenberg and Taghon,
2005; Blom and Spits, 2006), that are well conserved in the
promoter sequence of the mouse pvt-1 and rat mis-1 genes
(data not shown).
A previous report, showing that in mouse PVT-1 expression
is highest when IgL genes are rearranged or actively transcribed
during B-cell differentiation, further supports the importance of
this region in transcriptional regulation (Huppi et al., 1990).
Finally the strong activity observed with the plasmid containing
the genomic fragment from nt 296 to þ132 in LAN-5 cells,
which express high levels of N-Myc proteins due to gene
amplification, could be explained by the presence of two
non-canonical E-boxes (50 -CACGCG-30 ) located 48 bp apart in
the proximal PVT-1 promoter sequence (155/95). Such a
variant E-box has been found in the promoters of several
Myc target genes and it is bound efficiently by Myc/Max
heterodimers in vivo and in vitro (Zeller et al., 2001; Fernandez
et al., 2003). Furthermore a computer-assisted (e-Ensamble,
NCBI) alignment of the human PVT-1 promoter nucleotide
sequence with the homologous mouse and rat sequences,
indicates the existence of a significant evolutionary
conservation both at the nucleotide level and in the spacing of
the E-boxes relative to the transcriptional start site (Fig. 4A).
This observation strongly suggests a functional constraint
relative to the presence of these two putative Myc-binding sites
in the promoter region of the PVT-1 gene.
Fig. 4. PVT-1 transactivation by c-Myc. A: Evolutionarily conserved
E-box sites in the PVT-1 promoter region. Sequence alignment of the
elements conserved in human (Hu), mouse (Mo) and rat (Rt) is shown,
identical nucleotides are indicated by asterisks. The highly conserved
core of Myc-binding sites are boxed. Exon 1 start site has been defined
according to the human sequence as reported previously
(Shitivelman and Bishop, 1989). B: Asynchronous RAT1-MycER cells
were transiently transfected with the luciferase reporter plasmids
pGL3-basic, Pvt(S296/R132), Pvt(S296/R132) E-box1-mut,
Pvt(S296/R132) E-box2-mut and 4xE/tk-Luc, as described in
Materials and Methods. To allow translocation of MycER into the
nucleus cells were stimulated for 4 h with 4HOT (striped bar), a
parallel set of cells did not receive any treatment (black bar).
Luciferase activity is expressed relative to the activity of pGL3-basic
plasmid in untreated RAT1-MycER cells that was settled to 1
(mean W SD). C: Western blot analysis of c-Myc proteins in nuclear
extracts of RAT1-MycER cells before (S) and after (R) a 4 h treatment
with 4HOT.
c-Myc is a PVT-1 transcriptional activator
N-Myc binds to the PVT-1 E-boxes region in vivo
To test the hypothesis that the PVT-1 gene is a Myc target, we
performed transfection experiments using a RAT1 fibroblast
cell line that constitutively expresses MycER, a fusion protein
consisting of Myc and a mutated form of the hormone binding
domain of the oestrogen receptor (Littlewood et al., 1995).
When these cells are exposed to 4-hydroxytamoxifen (4OHT),
the ligand-bound MycER protein disengages from the
chaperone protein HSP90 in the cytoplasm and translocates to
the nucleus (see the Western blot in Fig. 4C), where it activates
or represses transcription of Myc target genes. As shown in
Figure 4B, the luciferase activity of the reporter carrying the
296/þ132 PVT-1 promoter region, containing the E-box1 and
E-box2 sequences (Fig. 4A), increased more than two fold in
cells treated with 4OHT, a similar increase was observed with
the reporter containing a mutated E-box1 (Pvt(296/þ132)Ebox1-mut) or the 4xE/TK-Luc plasmid, which contains four
canonical Myc binding sites (Mori et al., 1998). Such induction
was not observed when a reporter plasmid containing the
PVT-1 promoter with mutated E-box2 (Pvt(296/þ132)Ebox2-Mut) was used. These results indicate that the human
PVT-1 promoter displays a c-Myc-dependent regulation
mediated by the conserved E-box2 located 95 nt upstream of
the transcriptional start site.
The results obtained with the RAT1-MycER cells prompted us
to investigate the functional relationship between the N-Myc
protein(s) and the PVT-1 gene in neuroblastoma cells, where
the maximum activity of the reporter containing the putative
Myc binding site was observed (see Fig. 3B). Comparative
analysis of N-Myc and PVT-1 mRNA levels in two
neuroblastoma cell lines: LAN-5, carrying an amplified N-Myc
gene, and SK-N-SH, with a single copy N-Myc gene, showed a
consistent correlation between down-regulation of N-Myc
expression and decrease of PVT-1 transcripts upon retinoic
acid (RA)-induced differentiation of the cells (Fig. 5A).
Therefore, the putative interaction between the cis-regulatory
element in the PVT-1 promoter and N-Myc proteins expressed
in proliferating and differentiated LAN-5 neuroblastoma cells
(Fig. 5B), and the presence of acetylated histone H4 was
investigated in vivo by chromatin-immunoprecipitation (ChIP).
After formaldehyde-crosslinking, sheared chromatin was
immunoprecipated with anti-N-Myc, or anti-acetylated H4
antibodies, or pre-immune serum. PCR reactions were
performed using oligonucleotides specifically amplifying the
PVT-1 promoter region containing the two E-boxes (nt 220/
35); primers directed to a region of the alpha enolase (ENO1)
gene promoter, containing an E-box that has been shown to be
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
PVT-1 EXPRESSION IN NORMAL AND TRANSFORMED HUMAN CELLS
Fig. 5. Regulation of the PVT-1 gene by N-Myc. A: Expression of PVT-1 and N-Myc mRNAs in proliferating and differentiated neuroblastoma cells,
LAN-5 and SK-N-SH (1-, 4-, 8-day, retinoic acid treatment). To verify equal input of RNA oligonucleotide primers for GAPDH were included in each
PCR reaction. Total cellular RNA was extracted, reverse transcribed and amplified with gene specific primers as described in Materials and
Methods. B: Western blot analysis of N-Myc proteins in proliferating (0 days) or retinoic acid treated (4 and 8 days) neuroblastoma LAN-5 cell
extracts. The same blot was stripped and probed with anti-HSP70 antibodies as loading control. C: Chromatin immunoprecipitation (ChIP)
showing binding of N-Myc proteins to the PVT-1 promoter. Chromatin was isolated from proliferating (0 days) or retinoic acid treated (3 days)
LAN-5 neuroblastoma cells and immunoprecipitated using anti-N-Myc antibodies, anti-acetylated histone H4 (AcH4) or preimmune IgG. PCR was
performed on ‘‘input’’ DNA as well as on immunoprecipitated DNA using primers directed to PVT-1 promoter region containing the two noncanonical E-boxes (PVT-1), primers targeted to a region of the alpha enolase gene promoter containing an high affinity E-box (ENO1) and primers
directed to the PVT-1 exon I region (PVT-1/exon1), as described in Materials and Methods. D: Quantification of immunoprecipitated chromatin by
real-time PCR. The amount of immunoprecipitated DNA, amplified with the same gene-specific primers described in (C), was calculated relative
to that present in total input chromatin (% input), as described in Materials and Methods. Each data point represents the average of triplicates from
two independent ChIP experiments W SD.
a high affinity binding site of Myc proteins (Fernandez et al.,
2003), and primers set targeted to an unrelated region of the
PVT-1 gene (exon 1, nt þ113/þ267) as a negative control. As
shown in Figure 5C immunoprecipitates with anti-N-Myc and
anti-acetylated histone H4 both yielded PVT-1-specific and
ENO1-specific PCR products in proliferating and differentiated
cells (3 days in RA). No enrichment was observed when primers
for PVT-1 exon 1 were used. To gain quantitative informations
on the DNA enrichment obtained by immunoprecipitation with
specific antibodies, PCR reactions were performed using a
real-time system. As shown in Figure 5D, the amount of PVT-1
and ENO1 genomic DNA coprecipitated with N-Myc from
proliferating LAN-5 cells was about 0.3% of that present in total
input chromatin, and it was drastically reduced in differentiated
LAN-5 cells. A similar pattern of coprecipitation was observed
when anti-acetylated histone H4 antibodies were used. These
results indicate that N-Myc binds to the E-boxes region of the
PVT-1 promoter and the concomitant presence of acetylated
histone H4 is indicative of transcriptional activation.
Furthermore, differentiation by RA treatment causes a
decrease in binding activity and in the amount of acetylated
histone H4.
Conclusion
The current study shows that c-Myc and PVT-1 transcripts have
a different tissue distribution in physiological conditions
suggesting that the transcriptional regulation of these two genes
is not correlated in normal cells. Conversely, in cancer cells
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
PVT-1 expression level and c-Myc or N-Myc mRNA levels are
well correlated.
The human PVT-1 gene contains two non canonical
Myc-binding sites (E-box CACGCG) in the promoter region
proximal to the transcriptional start site (155/95) the
consensus and the surrounding sequences are conserved in the
homologous mouse and rat genes. Our transactivation studies
indicate that one of the two E-boxes is important for PVT-1
promoter transcriptional regulation by c-Myc proteins. ChIP
analysis in neuroblastoma cells indicates that the same region
containing the two E-boxes is bound in vivo by N-Myc proteins,
and this is associated with a local hyperacetylation of histone
H4. Furthermore, the amount of N-Myc proteins recruited to
the PVT-1 promoter and the amount of acetylated histone H4
decrease in differentiated cells correlating with decreased
levels of PVT-1 mRNA. These findings imply that the E-boxes
region in the PVT-1 proximal promoter is important for PVT-1
transcriptional regulation by Myc proteins and reveal a novel
cross-talk between PVT-1 and N-Myc in neuroblastoma cells.
The role of Myc as a transcription factor suggests that the
initial steps in Myc-induced tumorigenesis involve the
transcription of critical genes in the transformation process.
Several reports revealed that Myc affects transcription of a large
number of genes, as listed in the Myc target gene database
(www.myccancergene.org). However, it is still an open
question as to which of the reported target genes triggered by
ectopic Myc expression are indeed essential for tumorigenesis.
Isolation and characterization of full-length cDNAs for human
PVT-1 gene from different cell types will give new insights into
the coding potential of the gene and the possibility to set an
517
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CARRAMUSA ET AL.
efficient siRNA-based strategy for knocking-down the gene to
further explore the biological significance of its overexpression
in transformed cells.
Acknowledgments
We are grateful to H. Ariga and M. Grossi for kindly providing
p4xE/Tk-Luc plasmid and the RAT1-MycER cell line,
respectively. We thank Patrizia Rubino for excellent technical
assistance. This work was supported in part by grants from
Ministero dell’Università e della Ricerca (MIUR) and POR Sicilia
(misura 3.14-DiaMol) to S.F.
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