Structure, Expression, and Function of Human Pituitary Tumor-Transforming Gene (PTTG)
Xun Zhang,
Gregory A. Horwitz,
Toni R. Prezant,
Alberto Valentini,
Masahiro Nakashima,
Marcello D. Bronstein and
Shlomo Melmed
Cedars-Sinai Research Institute-UCLA School of Medicine
(X.Z., G.A.H., T.R.P., A.V., M.N., S.M.) Los Angeles, California
90048
Neuroendocrine Unit (M.D.B.) Division of Functional
Neurosurgery University of São Paulo Medical School
São Paulo, SP, Brazil 01406-100
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ABSTRACT
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Despite advances in characterizing the
pathophysiology and genetics of pituitary tumors, molecular mechanisms
of their pathogenesis are poorly understood. Recently, we isolated a
transforming gene [pituitary tumor-transforming gene (PTTG)] from rat
pituitary tumor cells. Here we describe the cloning of human PTTG,
which is located on chromosome 5q33 and shares striking sequence
homology with its rat counterpart. Northern analysis revealed PTTG
expression in normal adult testis, thymus, colon, small intestine,
brain, lung, and fetal liver, but most abundant levels of PTTG mRNA
were observed in several carcinoma cell lines. Stable transfection of
NIH 3T3 cells with human PTTG cDNA caused anchorage-independent
transformation in vitro and induced in vivo
tumor formation when transfectants were injected into athymic mice.
Overexpression of PTTG in transfected NIH 3T3 cells also stimulated
expression and secretion of basic fibroblast growth factor, a
human pituitary tumor growth-regulating factor. A proline-rich region,
which contains two PXXP motifs for the SH3 domain-binding site, was
detected in the PTTG protein sequence. When these proline residues were
changed by site-directed mutagenesis, PTTG in vitro
transforming and in vivo tumor-inducing activity, as well
as stimulation of basic fibroblast growth factor, was abrogated. These
results indicate that human PTTG, a novel oncogene, may function
through SH3-mediated signal transduction pathways and activation of
growth factor(s).
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INTRODUCTION
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Tumorigenesis is a multistep process, involving activation of
oncogenes, growth factors, and their receptors, or the inactivation of
tumor suppressor genes (1). Abnormal gene expression in tumor cells is
associated with several characteristics that differ from normal cells,
such as cell differentiation (2), DNA repair (3), cell-cell
communication (4, 5), cell-matrix interaction (6), tumor invasion,
motility and metastasis (7, 8), angiogenesis (9), and apoptosis (10).
Despite major advances in studying these characteristics, understanding
tumorigenesis as a continuous, multifunctional process is still
limited.
Pituitary tumors are well differentiated adenomas accounting for
10% of intracranial neoplasms, and clinically silent pituitary
microadenomas are encountered in up to 23% of unselected adult
autopsies (11, 12). These monoclonal adenomas are either nonfunctioning
and do not secrete pituitary trophic hormones or are functioning and
secrete one or more hormones including PRL, GH, ACTH, or rarely,
glycoprotein hormones. These hypersecretory syndromes are associated
with hypogonadism, infertility, acromegaly, Cushings disease, or
rarely, hyperthyroidism (13). Most pituitary tumors are histologically
benign. True pituitary carcinomas are extremely rare, and documentation
of distant metastasis is the sole diagnostic criterion for malignancy
(14).
Pituitary tumor pathogenesis has been extensively studied (15). Several
intrinsic mutations resulting in activation of tumor-promoting genes as
well as inactivation of tumor suppressor genes have been described,
including G protein (Gs
) mutations (16, 17), rarely
occurring ras mutations in invasive tumors (18, 19), loss of
heterozygosity involving the 11q13 region (20), loss of purine-binding
factor gene (nm23) expression (21), and, in mouse models, disruption of
RB (22) and cyclin-dependent kinase inhibitors (23, 24). However, only
G protein mutations have reproducibly been identified in a subset of
sporadic GH-secreting pituitary adenomas (25). The molecular etiology
of these tumors remains elusive, and other mechanisms may be invoked in
pituitary tumorigenesis. Recently, a novel pituitary tumor-transforming
cDNA (PTTG) was isolated in our laboratory from rat GH4 pituitary tumor
cells (26). Overexpression of rat PTTG resulted in cell transformation
in vitro and induced in vivo tumor formation in
athymic mice.
We now characterize the human pituitary tumor-transforming gene (PTTG).
It is abundantly expressed in malignant tumor cells, as well as in some
normal tissues, and potently transforms cells both in vitro
and in vivo. Overexpression of PTTG in transfected NIH 3T3
cells increases basic FGF mRNA level as well as stimulates its
secretion. As point mutations in a proline-rich region near the PTTG C
terminus abrogate its transforming ability and block basic FGF
production, PTTG function in tumorigenesis may involve intracellular
SH3 signals and growth factor production.
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RESULTS
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Molecular Cloning of Human PTTG
An open reading frame containing 609 bp was revealed in the
positive clones obtained from human fetal liver cDNA library using a
0.6-kb rat PTTG cDNA as screening probe (Fig. 1b
). The presence of an in-frame stop
codon before the predicted initiation codon suggests that it is
complete. The homology between DNA sequences of this open reading frame
and the coding region of rat PTTG is 85%. Amino acid sequence
comparison between the translated product of this cDNA and rat PTTG
protein reveals 77% identity and 89% homology. Since all positive
clones were sequenced and no other cDNA fragment with higher homology
was detected in the library, it appears that these cDNA clones
represent human homologs of rat PTTG.

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Figure 1. Human PTTG Genomic and cDNA Structure
a, Human PTTG genomic structure, with numbering according to the cDNA
sequence in panel b. The translation initiation (ATG) and stop (TAA)
codons in the cDNA are indicated. NT, Nontranslated region. b,
Nucleotide sequence of human PTTG cDNA. The translation initiation and
stop codons for the open reading frame are bold and
underlined. The in-frame stop codon upstream of the initiation
codon is bold and italicized. c, Amino acid sequence of
PTTG protein deduced from the coding region of cDNA. The basic amino
acid-rich domain is underlined, and the basic amino acid
residues are bold and italicized. The proline-rich
motifs are bold.
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GenBank search did not reveal a known protein with structural
similarity to PTTG. However, we detected a basic amino acid-rich region
near the N terminus (from position 58 to 101, with 32% basic amino
acid residues) and a proline-rich region near the C-terminus of human
PTTG protein (Fig. 1c
). This proline-rich region contains two PXXP
motifs consistent with the previously identified SH3-binding site (27).
The presence of these functional motifs suggests that human PTTG
protein may be involved in SH3 domain-mediated signal transduction
pathways (28, 29).
The human PTTG cDNA was used to screen a human genomic library, and
positive genomic clones were subjected to sequence analysis. The
results were compared with the cDNA sequence of human PTTG and revealed
four introns within the coding region (Fig. 1a
).
Chromosomal Localization of Human PTTG
DNA from the Stanford Human-Hamster G3 Radiation Hybrid Mapping
Panel was used as template in PCR reactions with PTTG-specific primers.
The amplified products were sequenced to confirm that they indeed
contain human PTTG sequences. By electronic analysis at the Stanford
Human Genome Center website, PTTG was localized to 21.01
centirads from the marker D5S2576 with a LOD score of
8.48. According to neighboring Genethon markers in the SCIENCE96
Transcription Map, this marker is most likely located within the
interval 5q3234 of chromosome 5.
This mapping result was further confirmed by fluorescence in
situ hybridization (FISH), using a 16-kb human genomic fragment
containing PTTG as a probe. The initial experiment resulted in specific
labeling of the distal long arm of a group B chromosome, which was
believed to be chromosome 5 based on its size, morphology, and banding
pattern. In a second experiment, a probe previously mapped to 5q21 was
cohybridized with PTTG, resulting in specific labeling of the middle
and distal long arm of chromosome 5, respectively (Fig. 2a
). Among a total of 80 metaphase cells
analyzed, 63 exhibited specific labeling, showing that human PTTG is
located at a position that is 84% the distance from the centromere to
the telomere of chromosome arm 5q, an area corresponding to band 5q33
according to the International System for Human Cytogenetic
Nomenclature 1995. Thus, the results concur and localize PTTG to 5q33
(Fig. 2b
).

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Figure 2. Chromosomal Localization as Determined by FISH
a, Metaphase chromosomes hybridized with human PTTG probe (arrow
a) and 5q21 control probe (arrow b). b, Ideogram
illustrating the chromosomal position of human PTTG at 5q33 according
to the International System for Human Cytogenetic Nomenclature 1995.
Arrow indicates human PTTG position.
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Tissue Distribution of Human PTTG mRNA
The expression pattern of human PTTG mRNA in normal human
adult and fetal tissues and in several human carcinomas is depicted in
Fig. 3
. A strong mRNA signal of
approximately 0.8 kb was detected in human fetal liver (Fig. 3b
). In
normal human adult tissues, abundant PTTG expression was evident in
testis. Strong expression was also observed in thymus, and weak
expression signals were seen in colon, small intestine, brain,
placenta, and pancreas (Fig. 3a
). Interestingly, when human malignant
tumor cells were tested, PTTG was found to be highly expressed in all
cell lines examined (Fig. 3c
). PTTG mRNA was also detected in several
human pituitary tumors, including nonfunctioning, PRL-secreting, and
ACTH-secreting tumors (Fig. 3d
). No mutations of the PTTG-coding region
in tumors were detected by RT-PCR followed by sequence analysis (data
not shown).

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Figure 3. Tissue Distribution of Human PTTG mRNA
Two micrograms of poly A+ RNA derived from the indicated
tissues were loaded in each lane. Top panel shows result
of Northern hybridization to PTTG cDNA probe, and bottom
panel shows hybridization to ß-actin probe (control). a,
Normal human adult tissues. b, Normal human fetal tissues. c, Human
cancer cells. From left to right: promyelocytic leukemia
HL-60; HeLa cell S3; chronic myelogenous leukemia K-562;
lymphoblastic leukemia MOLT-4; Burkitts lymphoma Raji; colorectal
adenocarcinoma SW480; lung carcinoma A549; melanoma G361. d, Pituitary
tumors. NF, Nonfunctioning tumor; PRL, PRL-secreting tumor; ACTH,
ACTH-secreting tumor.
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Effects of Human PTTG Overexpression on Cell Transformation and
Tumor Induction
Since a GeneBank search revealed no known proteins structurally
similar to PTTG, its role in tumor formation is as yet unclear.
However, the presence of a proline-rich region containing PXXP motifs
near the C terminus of PTTG protein suggests that it may be involved in
SH3-mediated intracellular signal transduction pathways. To explore the
function of this domain and its relationship to PTTG-transforming
ability, we constructed human PTTG mutants by PCR-based site-directed
mutagenesis. The following amino acid residues in the SH3-binding
motifs were mutated: P163A, P170L, P172A, and P173L. Mutant cDNA, as
well as wild-type PTTG cDNA, was cloned into the mammalian expression
vector under control of the cytomegalovirus (CMV) promoter and stably
transfected into NIH 3T3 cells. Overexpression of wild-type and mutant
PTTG in each transfected cell line was confirmed by Northern analysis,
RT-PCR followed by direct sequence analysis, and Western blot (Fig. 4
). The point mutations did not change
protein expression or susceptibility to proteolysis, since PTTG protein
with the same molecular weight was expressed in each transfected cell
type at similar levels, as shown by Western blot (Fig. 4c
). The
transforming ability of these cells was tested in an
anchorage-independent growth assay. We found that NIH 3T3 cells
overexpressing PTTG formed large colonies (numbers ranged from 198
± 6 to 267 ± 23 colonies per plate, mean ± SD)
on soft agar, while control NIH 3T3 cells containing the same
expression vector but lacking PTTG cDNA insert did not induce
significant colony foci under the same conditions (22 ± 1
colonies per plate) (Table 1
). In
contrast, the number and size of colonies formed from NIH 3T3 cells
expressing mutant PTTG were greatly reduced (ranging from 57 ± 6
to 60 ± 5 colonies per plate) (Table 1
and Fig. 5
). Furthermore, when these cells were
injected subcutaneously into athymic nude mice, PTTG-overexpressing
cells caused tumor formation within 2 weeks in all five injected
animals (tumor weights ranged from 560 to 1000 mg) (Fig. 6
). In five mice injected with control
transfectants (expression vector alone), only one developed a much
smaller tumor weighing only 100 mg. As expected, when cells expressing
mutant PTTG were injected into nude mice, no tumor formation was
observed even after 3 weeks of injection (Fig. 6
), consistent with the
results obtained in the anchorage-independent growth assay. These
results thus demonstrate the in vitro transforming activity
and in vivo tumor-inducing potential of human PTTG and also
strongly suggest that signaling protein(s) containing SH3 domain(s)
mediate PTTG action.

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Figure 4. Wild-Type and Mutant PTTG Expression in Transfected
NIH 3T3 Cells
a, A representative Northern blot in which 20 µg total RNA from each
cell line were used to hybridize with human PTTG cDNA probe (top
panel) or ß-actin probe (bottom panel). b, A
representative sequencing gel from RT-PCR followed by direct sequence
analysis showing wild-type PTTG and mutant PTTG expression in a
respective transfectant. The arrows point to nucleotide
changes. A silent mutation (*) was introduced to obtain equal melting
points for the different primers. c, A representative Western blot in
which 40 µg protein extracted from each transfectant were analyzed
using a purified anti-PTTG polyclonal antibody. C, Cell lines
transfected with vector alone; WT, cell lines transfected with
wild-type PTTG expression vector; M, cell lines transfected with mutant
PTTG expression vector.
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Figure 5. Colony Formation of NIH 3T3 Cells Transfected with
Wild-Type and Mutant PTTG Expression Vector on Soft Agar
a, Cells transfected with vector pCI-neo alone. b, Cells transfected
with vector pCI-neo containing wild-type PTTG cDNA. c, Cells
transfected with vector pCI-neo containing PTTG cDNA mutated in the
proline-rich region (P163A, P170L, P172A, and P173L). d, High
magnification of colonies formed from cells transfected with vector
pCI-neo containing wild-type PTTG cDNA.
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Figure 6. Tumor Formation Induced by Human PTTG-Expressing
Cells in Nude Mice
Each mouse was injected subcutaneously with 3 x 105
control, wild-type PTTG-overexpressing, or mutant PTTG-overexpressing
cells. After 2 weeks, mice were photographed and killed, and their
tumors were excised and weighed. Vector, Cell line transfected with
vectors only; wt PTTG, cell line transfected with wild-type human PTTG
expression vectors; mPTTG, cell line transfected with mutant human PTTG
expression vector; None, no detectable tumor. The mouse on the
left was injected with control cells transfected with
vector only. The mouse in the middle was injected with
cells transfected with wild-type PTTG expression vector. The mouse on
the right was injected with cells transfected with
mutant PTTG expression vector.
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Stimulation of Basic FGF Expression and Secretion by Human PTTG
As PTTG-expressing NIH 3T3 cells were able to induce solid
tumor growth in vivo, PTTG may activate growth factor and/or
angiogenesis pathways. To test this hypothesis, we examined the
expression of two important angiogenic factors, vascular endothelial
growth factor (VEGF) and basic fibroblast growth factor (bFGF), in the
PTTG-transfected NIH 3T3 cells. Northern analysis showed that, although
no difference in VEGF mRNA expression was found in control and
PTTG-transfected cells (data not shown), bFGF mRNA levels in
PTTG-transfected cells were increased compared with mock-transfected
control cells (Fig. 7a
). In the cells
transfected with mutant PTTG, the abundance of bFGF mRNA transcripts
was low, similar to that observed in the control cells.

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Figure 7. Human PTTG Stimulates bFGF Production
a, A representative Northern blot in which 20 µg total RNA from each
cell line were used to hybridize with human bFGF cDNA probe (top
panel) or ß-actin probe (bottom panel). b,
bFGF concentration in conditioned medium derived from each PTTG
transfectant cultured for 72 h as measured by ELISA. C, Cell lines
transfected with vector alone; WT, cell lines transfected with
wild-type PTTG expression vector; M, cell lines transfected with mutant
PTTG (P163A, P170L, P172A, and P173L) expression vector.
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To further confirm bFGF regulation by PTTG at the protein level,
enzyme-linked immunosorbent assay (ELISA) was performed to examine bFGF
levels in conditioned culture medium. As shown in Fig. 7b
, bFGF levels
were markedly higher in the conditioned medium collected from wild-type
human PTTG transfectants cultured for 72 h than those from control
and mutant transfectants. Differences of total protein concentrations
in these cultures were less than 10%. Therefore, PTTG not only
enhances bFGF mRNA levels, but also stimulates bFGF secretion. The PTTG
proline-rich region containing two SH3-binding motifs appears critical
for this function.
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DISCUSSION
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We have isolated a human gene that shows a striking structural
similarity (85% identity for cDNA and 89% similarity for amino acid
sequences) to rat PTTG. Human PTTG is located on chromosome 5, at
position 5q33. Like its rat counterpart, it induces cell transformation
in vitro and in vivo.
In normal adult rat tissues, PTTG expression is restricted to the
testis (26). The normal tissue distribution of human PTTG is also
limited; however, it is not restricted to testis but is also expressed
in thymus, colon, small intestine, and, weakly, in brain and placenta,
indicating that it may also play a role in certain normal cellular
functions. The expression of human PTTG in fetal liver, but not in
adult liver, suggests that it is differentially regulated and possibly
functions during development. Interestingly, PTTG is highly expressed
in all carcinoma cell lines tested, suggesting that it is a common and
important factor for most malignant tumor types.
The chromosomal location of human PTTG, 5q33, is associated with
reports of recurrent neoplastic abnormalities, including myeloid
leukemia, chronic myeloproliferative disorder, myelodysplastic
syndrome, squamous cell carcinoma, and lipoma (30). Using RT-PCR and
direct sequencing, we examined PTTG in several human carcinoma cell
lines, including cervix carcinoma HeLa cell, choriocarcinomas JEG-3 and
JAR, breast adenocarcinoma MCF-7, osteogenic sarcoma U-2 OS,
hepatocellular carcinoma Hep 3B, lung carcinoma EY, and thyroid
carcinoma TC-1. Although no mutation was detected within the PTTG
coding region, PTTG expression levels were high in all carcinoma cell
lines tested. Thus, putative mutations in the gene-regulatory regions
may cause PTTG dysregulation, and enhanced PTTG expression may mediate
neoplastic cell transformation. We also found that PTTG mRNA was
expressed in several pituitary tumors, indicating that it may be
involved in pituitary tumorigenesis. Interestingly, PTTG mRNA levels in
benign pituitary tumors, in general, were much lower than in malignant
carcinomas (31). Therefore, although it seems that tumor PTTG
expression correlates with malignancy, further confirmation is
needed.
The transforming ability and high level of PTTG expression in malignant
tumors demonstrate its involvement in tumorigenesis, although
mechanisms of PTTG action and its relationship to other oncogene or
tumor suppressor gene products is yet unclear. The presence of a basic
amino acid-rich region and a proline-rich region in PTTG protein
provide insights regarding its function. Basic amino acid-rich domains
have been suggested as a nuclear localization signal (31, 32, 33), although
the subcellular localization of PTTG is still undetermined. The
proline-rich region of the human PTTG protein contains two PXXP motifs,
which are potential binding sites for SH3-domains (27), important
mediators of intracellular signal transduction (28, 34). Several
proteins have recently been identified to contain PXXP motifs and bind
to SH3 domains, such as GDP/GTP exchange factor SOS (35, 36), protein
kinases JNK and phosphatidylinositol 3-kinase (37, 38), and the
Abl oncogene product (39, 40). We report here that point mutations in
the PXXP motif of human PTTG abrogated its transforming and
tumor-inducing activity, despite expression of PTTG mRNA and protein in
these mutants. Thus, this PXXP motif is important for PTTG-mediated
transformation. Alternatively, another mechanism for the function of
this region could involve serine phosphorylation of adjacent regions
(41).
Interestingly, PTTG induces bFGF production at both the mRNA and
protein levels. bFGF is a major activating factor for angiogenesis (42, 43), a necessary process for the expansion of the primary tumor mass as
well as tumor metastasis (9, 44, 45). This is also in concurrence with
our finding that transfected cells overexpressing PTTG cause formation
of solid tumors in nude mice. Increased bFGF expression has been
reported in several human tumors, such as pancreatic carcinoma (46),
endometrial adenocarcinoma (47), advanced renal cell carcinoma (48),
primary breast cancer (49), and pituitary tumors (50), in which it is
considered a stimulating growth factor (51). Considering that the bFGF
receptor is a protein tyrosine kinase and that PTTG increases bFGF
production, we propose that one of the mechanisms of tumorigenesis by
PTTG involves an SH3 protein interacting with PTTG, stimulating gene
transcription and secretion of bFGF, and resulting in cell
transformation and tumor formation. This hypothesis is supported by our
observation that mutations of the proline-rich region, the potential
SH3-binding site, abrogate bFGF production as well as cell
transformation. These results establish human PTTG as a potent
tumor-promoting factor whose functions involve bFGF production and cell
transformation.
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MATERIALS AND METHODS
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Library Screening
A human fetal liver cDNA library (CLONTECH, Palo Alto, CA) was
screened as described (51), using a 0.6-kb radioactively labeled cDNA
fragment containing the entire rat PTTG coding region as a probe. The
cDNA inserts from positive clones were subcloned into plasmid
pBluescript-SK (Stratagene, La Jolla, CA) and subjected to sequence
analysis using a Sequenase kit (United States Biochemical Corp.,
Cleveland, OH) and SequeGel System (National Diagnostics, Atlanta,
GA).
A human genomic library (Stratagene, La Jolla, CA) was screened
according to the manufacturers protocol, using the radioactively
labeled human cDNA clone containing the complete coding region as a
probe. DNA from each positive phage clone was purified using Lambda DNA
preparation kit (Qiagen, Valencia, CA) and sequenced with a thermocycle
sequencing kit (Amersham, Arlington Heights, IL) and SequaGel System
(National Diagnostics).
Determination of Human PTTG Chromosomal Location
Chromosomal localization of human PTTG was determined by PCR
analysis of the Stanford Human-Hamster G3 Radiation Hybrid (RH) Mapping
Panel (Research Genetics, Huntsville, AL) as well as by FISH. DNA from
83 samples in the G3 RH mapping panel were amplified in 20-µl
reactions containing 50 ng DNA, 1.75 U Expand High Fidelity enzyme
(Boehringer-Mannheim, Indianapolis, IN), 1x reaction buffer with 1.5
mM MgCl2, 0.2 mM deoxynucleoside
triphosphates (dNTPs), and 300 nM human PTTG-specific
primers, 5'-CTGCCT-CAGATGATGCCTATCCAG-3'and
5'-CAAGCTCTCTCTCCTCGTCAAGG-3'. The PCR reactions were performed for
35 cycles of 94 C, 15 sec; 60 C, 30 sec; 68 C, 2.5 min. PCR products
were visualized in 1% agarose gels stained with ethidium bromide and
scored as positive ("1") if a strong (
3.5-kb PCR product) was
visualized, negative ("0") if the band was not visualized, or
ambiguous if a weak band was observed. The same results were obtained
when the panel was again amplified with GIBCO-BRL (Gaithersburg, MD)
Taq DNA polymerase. These data were submitted electronically
to the Stanford Human Genome Center website to determine linkage to
previously mapped markers. The FISH was performed at GenomeSystem, Inc.
(St. Louis, MO). Briefly, a 16-kb human genomic fragment containing
PTTG was labeled with digoxigenin dUTP by nick translation. Labeled
probe was combined with sheared human DNA and hybridized to normal
metaphase chromosomes derived from phytohemagglutinin-stimulated
peripheral blood lymphocytes. Specific hybridization signals were
detected by incubating the hybridized slides in fluoresceinated
antidigoxigenin antibodies followed by counterstaining with
4,6-diamidino-2-phenylindole (DAPI).
Northern Blot Analysis
RNA blots (CLONTECH, Palo Alto, CA) derived from normal human
adult and fetal tissue, as well as from malignant tumor cell lines and
fresh pituitary tumor specimens, were hybridized with a 0.7-kb
radiolabeled human cDNA fragment containing the complete coding region.
Northern blot analysis was performed using ExpressHyb Solution
(CLONTECH) or QuickHyb Solution (Stratagene, La Jolla, CA) according to
the manufacturers protocol.
Western Blot Analysis
An antirat PTTG polyclonal antibody was developed as described
previously (26). This antibody was purified by a PTTG affinity column
that couples 10 mg synthetic antigenic peptide with HiTrap
N-hydroxysuccinimide-activated column (Pharmacia,
Piscataway, NJ) according to manufacturers protocol. The affinity
column was washed with 75 mM Tris-HCl, pH 8.0, until no
protein appeared in the eluent. The purified antibody was eluted with
0.1 M glycine, 0.5 M NaOH, pH 2.7, and
neutralized with each volume of 2 N Tris-HCl, pH 8.0.
Western blot was performed as described previously (26) using this
purified antibody (1:100 dilution).
Stable Transfection of Human PTTG into NIH 3T3 Cells
The complete coding region of human PTTG cDNA was subcloned in
frame into mammalian expression vectors pBK-CMV (Stratagene) or pCI-neo
(Promega, Madison, WI) and transfected into NIH 3T3 fibroblast cells
with Lipofectamine (GIBCO-BRL) according to the manufacturers
protocol. Twenty-four hours after transfection, cells were serially
diluted and grown in selection medium containing 1 mg/ml G418 for 2
weeks. Individual clones were isolated and maintained in selection
medium. Total RNA was isolated from human PTTG-transfected cell lines
as well as from control cells in which the vector pBK-CMV or pCI-neo
had been transfected. Overexpression of human PTTG in each transfected
cell line was confirmed by Northern blotting.
Site-Directed Mutagenesis
Point mutations on the proline-rich domain of PTTG peptide were
generated by PCR-based site-directed mutagenesis. Two synthetic
oligonucleotides, 5'-GATGCTCTCCGCACTCTGGGAATCCAATCTG-3' and
5'-TTCACAAGTTGAGGGGCGCCCAGCTGAAACAG-3', which would cause amino acid
changes P163A, P170L, P172A, and P173L, were used to amplify human PTTG
cDNA cloned into pBlueScript-SK vector (Stratagene). The amplified cDNA
containing these mutations was then cloned into pCI-neo (Promega) and
used in stable transfection. Expression of mutated PTTG product in
transfected cells was confirmed by Northern analysis and RT-PCR
followed by direct sequence analysis.
In Vitro and in Vivo Transformation
Assay
Control and human PTTG-transfected cells were tested for
anchorage-independent growth in soft agar (53). Three milliliters of
soft agar (20% 2x DMEM, 50% DMEM, 10% FBS, and 20% 2.5% agar,
melted and mixed at 45 C) were added to 35-mm tissue culture dishes.
Ten thousand cells were mixed with 1 ml soft agar and added to each
dish. Cells were incubated for 2 weeks before colonies were counted and
photographed. For in vivo transformation, 1 x
105 control or human PTTG-transfected cells were
resuspended in 400 µl PBS and injected subcutaneously into nude mice
(five mice for each group). After two weeks, animals were photographed
and tumors were excised and weighed.
Hybridization with VEGF and bFGF cDNA Probes
The cDNA probes for VEGF and bFGF were generated by RT-PCR using
specific primers according to the published sequences (54, 55). These
cDNAs generated from PCR were cloned and confirmed by sequence
analysis. Total RNAs from cultured cells were extracted using Trizol
Reagent (GIBCO-BRL) and used in Northern analysis as described
previously (56).
ELISA of bFGF in Conditioned Medium
bFGF concentration in cell culture medium was assayed using
Quantikine HS Human FGF Basic Immunoasssay Kit (R&D Systems,
Minneapolis, MN) according to the manufacturers protocol. Cells
(1 x 105) were plated in 100-mm cell culture dishes.
After 72 h, the culture medium was collected, and 1 ml was
lyophilized and resuspended in 200 µl PBS for ELISA assay.
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ACKNOWLEDGMENTS
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We thank Drs. S. G. Ren and X. Li for technical help.
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FOOTNOTES
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Address requests for reprints to: Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu
This work was supported by NIH Grant DK-50238 (S.M.), Institutional
Training Grant DK-7682 (X.Z.), and the Doris Factor Molecular
Endocrinology Laboratory.
Received for publication July 23, 1998.
Revision received October 5, 1998.
Accepted for publication October 8, 1998.
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