Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 745-750
doi:10.1111/j.1745-7270.2007.00337.x
Overexpression of PTEN
Induces Cell Growth Arrest and Apoptosis in Human Breast Cancer ZR-75-1 Cells
Xiangyong LI1,
Guanping LIN1, Binhua
WU1,
Xin ZHOU2,
and Keyuan ZHOU1*
1 Institute
of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang
524023, China;
2 Center
of Clinical Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan
430071, China
Received: April 3,
2007
Accepted: June 18,
2007
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30672741), the Science Planning Foundation of Guangdong Province (No.
2005B10401011), and the Special Funds for Major Subject Program of Guangdong
Province (No. 9307)
*Corresponding
author: Tel, 86-759-2388301; Fax, 86-759-2284104; E-mail, [email protected]
Abstract Phosphatase and tensin homolog (PTEN) is a tumor
suppressor gene located at human chromosome 10q23, might play an important role
in cell proliferation, cell cycle and apoptosis of cancer cells. In this study,
the eukaryotic expression vectors pBP-wt-PTEN (containing a wild-type PTEN
gene) and pBP-G129R-PTEN (containing a mutant PTEN gene) were
used to transfect breast cancer ZR-75-1 cells. After transfection, ZR-75-1
cells expressing PTEN were obtained and tested. The blue exclusion assay
showed the growth rate of the cells transfected with pBP-wt-PTEN was
significantly lower than that of the control cells transfected with pBP-G129R-PTEN.
Analysis of the cell cycle by flow cytometry showed that the progression from
the G1 to the S phase was arrested in cells expressing wild-type PTEN.
Some typical morphological changes of apoptosis were also observed in cells
transfected with pBP-wt-PTEN, but not in those transfected with
pBP-G129R-PTEN. This study shows that overexpression of PTEN in
ZR-75-1 cells leads to cell growth arrest and apoptosis.
Keywords PTEN; tumor suppressor gene; breast
cancer; cell growth; apoptosis
PTEN is a tumor suppressor gene located
at human chromosome 10q23 that encodes a dual substrate-specific phosphatase.
This gene is frequently deleted or mutated in a wide range of human tumors and
tumor cell lines [1–3]. Previous studies have shown that transient expression of PTEN
in PTEN-null endometrial, melanoma and lymphoid cancers could suppress
cell growth and cause cell apoptosis [4,5]. Germline mutations of the PTEN
gene, including homozygous deletions, have been found in patients with Cowden’s
disease, an autosomal dominant syndrome carrying elevated risk for cancers of
the breast and thyroid [6,7]. Mutations of this gene were reported in two of 26
breast cancer cell lines and in two of 14 primary breast tumors examined [8],
indicating that loss of PTEN function is probably associated with
progression of breast cancers. However, whether overexpression of PTEN
in human breast cancer cells affects their growth and apoptosis remains to be
investigated.In the present study, we transfected the PTEN-null breast
cancer cell line ZR-75-1 with a PTEN-expressing vector and examined the
effect of PTEN overexpression on cell proliferation and apoptosis.
Materials and Methods
Cell culture
and transfection
The breast cancer cell line ZR-75-1 was obtained from the Chinese
Academy of Medical Sciences (Beijing, China). The breast cancer cells were
cultured in RPMI 1640 complete culture medium (GIBCO, New York, USA)
supplemented with 15% heat-inactivated fetal bovine serum (FBS; GIBCO),
benzylpenicillin (100 kU/L), and streptomycin (100 mg/L) at 37 ?C in a
humidified incubator, with 5% CO2 in air. The cells were
routinely passaged every 1 or 2 d. For transfection, 5?106 cells mixed with 500 ml of RPMI 1640 (supplemented
with 15% heat-inactivated FBS, without antibiotics) were seeded into a 24-well
plate and incubated at 37 ?C for 48 h. Nearly confluent cells were then
co-transfected with pBP-wt-PTEN plasmid or pBP-G129R-PTEN plasmid
(gifts of Prof. Frank FURNARI, Ludwig Institute for Cancer Research, San Diego,
USA) (2.0 mg/well) and pTR-UF5 plasmid (gift of Dr. Nicholas MUZYCKA, Florida
University, Gainesville, USA) harboring green fluorescence protein reporter
gene (0.2 mg/well) using a Lipofectamine 2000-mediated (Invitrogen, Carlsbad,
USA) method according to the manufacturer’s protocol (ratio of plasmid to
Lipofectamine was 1:1). Six hours after transfection, the medium was changed to
normal culture medium. Cells were continuously cultured until harvest for analysis.
Semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted 48 h after transfection using Trizol reagent
(GIBCO). RT was carried out using a one-step RT-PCR kit (Qiagen, Hilden,
Germany). The primers for PTEN (amplified products were 373 bp) were 5-aaagctggaaagggacgaac-3 (forward), and
5-caggtaacggctgagggaac-3
(reverse). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
served as an internal standard. The upstream primer of GAPDH (amplified
products were 226 bp) was 5-gaaggtgaaggtcggagtc-3,
and the downstream primer was 5-gaagatggtgatgggatttc-3.
Thermal cycle conditions were as follows: 50 ?C for 30 min, 95 ?C for 15 min,
followed by 30 cycles of 94 ?C for 45 s, 55 ?C for 1 min, and 72 ?C for 1 min,
with a final extension at 72 ?C for 10 min. The PCR products were
electrophoresed on 1% agarose gel, stained with ethidium bromide, and detected
by ultraviolet irradiation.
Western blot analysis
The cells were washed three times with cold phosphate-buffered
saline (PBS) 48 h after transfection, collected by scraping and lysed in 150 ml ice-cold Tris
buffer (50 mM, pH 8.0) containing 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% sodium
dodecyl sulfate (SDS), 1.0 mg/L aprotinin, and 0.2 mg/L phenylmethylsulphonyl
fluoride for 10 min. The extracts were centrifuged at 12,000 g for 15 min, and the concentration of protein in each lysate was
determined with Coomassie Brilliant Blue G-250. Loading buffer was added to
each lysate, which was subsequently boiled for 3 min and elestrophoresed on a
SDS-polyacrylamide gel. Before electrophoresis, the proteins were mixed with 2?loading buffer (containing 100 mM Tris-HCl, pH 6.8, 20% glycerin, 4%
SDS, 0.05 g/L tetrabromophenol sulfonphthalein, and 10% 2-b-mercaptoethanol)
by the same volume. Proteins were transferred onto nitrocellulose membranes
(Sigma, New York, USA). After blocking with 5% skim milk in Tris-HCl (pH 7.5)
at room temperature for 2 h, the nitrocellulose membranes were reacted for 2 h
with specific antibodies in the same blocking solution [PTEN antibody and
anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, USA) were used at
1:300 and 1:500 dilutions, respectively]. After extensive washing with Tris-HCl
containing 0.05% Tween 20, the membranes were reacted with anti-mouse
immunoglobulin G-horseradish peroxidase for PTEN protein detection. Finally,
protein bands were visualized using an electrochemiluminescence test kit (Santa
Cruz Biotechnology). Densitometric analyses were carried out using Scion Image
software Version Cot33 (Scion, Frederick, USA).
Cell growth assay
Cell growth was measured by Trypan blue staining. An equal number of
cells were plated into 12-well plates to be transfected with pBP-wt-PTEN
plasmid or pBP-G129R-PTEN plasmid and cultured for various times. After
incubation, the medium was removed. Cells were then washed with
phosphate-buffered NaCl solution and fixed with 12.5% glutaraldehyde for 20 min
at room temperature. Cells were rinsed with distilled water and incubated with
1 mg/ml Trypan blue (Sigma) for 30 min. Cells were then observed and counted
under an inverted microscope and growth curves were drawn using the cultured
time as abscissa and living cells as ordinate.
Fluorescence microscopy
ZR-75-1 cells (1?105) were seeded in a 35 cm Petri dish and cultured in RPMI 1640
supplemented with 15% heat-inactivated FBS without antibiotics. After
incubation at 37 ?C for 24 h, cells of two dishes were transfected with pBP-wt-PTEN
plasmid or pBP-G129R-PTEN plasmid (the final concentration of plasmid
was 2.0 mg/ml). After transfection for 6 h, the medium was changed to
complete culture medium. Cells were collected 24 h after the transfection and
centrifuged for 5 min at 200 g to remove cell debris, and cells were
washed three times with 0.9% saline after centrifugation for 5 min at 1000 g
to remove culture medium. Then 10 ml of 10 mg/L Hoechest 33258 was added to the
cell suspension and cells were incubated for 30 min in the dark. Ten
microliters of the stained cell suspensions was taken out, dripped on slides
and covered with a cover slip. The morphological changes of nuclei in pBP-wt-PTEN-transfected
ZR-75-1 cells were observed using fluorescence microscope (400?) to discriminate normal cells, apoptotic cells and necrotic cells.
Cells were photographed and the images were processed with Adobe Photoshop
software version 7.0 (Adobe, San Jose, USA).
Flow cytometry analysis
After treatment with pBP-wt-PTEN plasmid or pBP-G129R-PTEN
plasmid for 48 h, ZR-75-1 cells were harvested by centrifugation at 200 g
for 5 min to remove cell debris, and washed three times with PBS by
centrifugation at 1000 g for 5 min to remove culture medium. The cell
suspension was fixed in ice-cold 70% ethanol in PBS, and stored at 20 ?C. Prior
to analysis, the cells were washed and resuspended in PBS, and incubated with 1
g/L of RNase I and 20 g/L propidium iodide at 37 ?C for 30 min. Apoptosis was
analyzed with a flow cytometer (Coulter Becton Dickinson, Miami, USA). For each
sample, at least 1?104 cells
were analyzed by flow cytometry, and the percentage of apoptotic cells in the
sub-G1 phase was calculated using Multicycle software (Phoenix Flow
Systems, San Diego, USA).
Statistical analysis
All statistical analyses were carried out using one-way anova tests. Values of P<0.05 were considered significant.
Results
Overexpression of PTEN
in transfected cells
To verify the expression status of PTEN in the ZR-75-1 cells
transfected with pBP-wt-PTEN and pBP-G129R-PTEN, the mRNA and
protein levels of PTEN in the transfected cells were determined by
RT-PCR and Western blot analysis, respectively. RT-PCR analysis showed that PTEN
mRNA was abundant in cells transfected with either pBP-wt-PTEN or
pBP-G129R-PTEN, but undetectable in the untreated cells (Fig. 1).
Accordingly, an appreciable amount of PTEN protein was found in cells
transfected with pBP-wt-PTEN or pBP-G129R-PTEN, but not in the
untreated (control) cells (Fig. 2). These data clearly indicate that
transfection with pBP-wt-PTEN or pBP-G129R-PTEN resulted in
overexpression of PTEN in ZR-75-1 cells.
Effect of PTEN
overexpression on cell growth
To investigate whether increased levels of PTEN in ZR-75-1 affect
cell growth, the number of cells at each time point after transfection was
determined by Trypan blue staining. Compared with untreated cells, the cells
transfected with pBP-wt-PTEN (expressing wild-type PTEN) showed a
significantly lower growth rate, whereas the cells transfected with pBP-G129R-PTEN
(expressing mutant PTEN) showed no difference in their growth rate (Fig.
3). Flow cytometry analysis indicated that there were a significant
increase in the number of cells at the G1 phase
and a decrease in the number of cells at the S phase in the pBP-wt-PTEN-transfected
cells, but not in the pBP-G129R-PTEN-transfected cells, suggesting that
overexpression of wild-type PTEN could induce G1 arrest
in ZR-75-1 cells.
Effect of PTEN
overexpression on cell apoptosis
To examine whether overexpression of PTEN
could induce cell apoptosis, detection of apoptotic cells using Hoechst
33258 staining and flow cytometry was carried out after transfection of ZR-75-1
cells with pBP-wt-PTEN or pBP-G129R-PTEN. As shown in Fig. 4,
there were very few apoptotic cells in the untransfected and pBP-G129R-PTEN-transfected
cells (expressing mutant PTEN). In contrast, many more apoptotic cells,
characterized by apoptotic bodies and fragmentation of nuclei, were observed in
the pBP-wt-PTEN-transfected cells (expressing wild-type PTEN).
Flow cytometry analysis showed that approximately 7% of pBP-wt-PTEN-transfected
cells were apoptotic (Fig. 5). These results suggest that overexpression
of wild-type PTEN in ZR-75-1 cells could induce apoptosis.
Discussion
Recently, with the development of molecular biology and its
application in oncology, it has been recognized that the activation of
oncogenes or the inactivation of cancer suppressor genes plays a great role in the
development and progression of cancer. According to published studies, somatic
mutations or deletion of cancer suppressor genes such as TP53 and p21
were closely correlated to the occurrence and development of tumor [9]. PTEN
is a major tumor suppressor gene identified on human chromosome 10q23 which
encodes a protein of 403 amino acids that includes a phosphatase core motif and
two potential tyrosine phosphate acceptor motifs. It is considered as a
candidate tumor suppressor gene based on the finding that mutation or loss of
this gene has been linked to a variety of common human cancers, including
breast, prostate, and brain cancer. PTEN is frequently deleted or
mutated in a wide range of human tumors and tumor cell lines such as
glioblastoma and melanoma, and lymphoid, lung, and endometrial cancers.
Furthermore, germline PTEN mutations have been found in patients with
juvenile polyposis coli, Cowden’s disease, a multiple hamartoma syndrome with a
high risk of breast and thyroid cancer, and the related hamartomatous polyposis
syndrome, Cowden’s syndrome [10], suggesting that inactivation of PTEN
plays an important role in tumorigenicity.In this study we have examined the effect of overexpression of PTEN
on the growth and apoptosis of PTEN-null ZR-75-1 cells. Our results have
shown an obvious correlation between the number of apoptotic cells, observed by
Hoechst 33258 staining and flow cytometry analysis, and the increased level of PTEN
mRNA, determined by semiquantitative RT-PCR and Western blot analysis. The
change in the mRNA and protein levels in pBP-wt-PTEN-transfected cells
was obvious, and apoptotic cells were also observed in the same group of cells
48 h after transfection. This strongly suggests that overexpression of PTEN
by transfection is responsible for the apoptosis in ZR-75-1 cells. The data
from semiquantitative RT-PCR and Western blot analysis also show a parallel or
corresponding change between PTEN mRNA and PTEN protein in the pBP-wt-PTEN-transfected
cells. Furthermore, our flow cytometry results also show that overexpression of
PTEN in ZR-75-1 cells caused growth suppression mediated initially by G1 arrest, followed by cell death, in agreement with previous reports
for other cancers [11].In the present study, both pBP-wt-PTEN plasmid and a
phosphatase-inactivating type plasmid pBP-G129R-PTEN were used to
transfect cells to provide a helpful control. According to our results,
overexpression of PTEN was found in the pBP-G129R-PTEN-transfected
group, but apoptotic cells were nearly absent, and no detectable change in the
cell cycle was observed compared with the untreated group, indicating that the
phosphatase activity of PTEN is required for the inhibition of cell
growth and induction of apoptosis [12,13].Our finding that overexpression of PTEN induced cell growth
arrest and apoptosis in the ZR-75-1 cell line suggests a role for PTEN
as a tumor suppressor gene in the prevention and treatment of breast cancer.
Our data also support the notion that PTEN might be a new target for
cancer gene therapy.
Acknowledgements
We thank Prof. Frank FURNARI and the Ludwig
Institute for Cancer Research (San Diego, USA) for help with pBP-wt-PTEN
and pBP-G129R-PTEN eukaryotic expression vectors. We also thank Dr.
Jingxuang Kang (Department of
Medicine, Massachusetts General Hospital and Harvard Medical School, Boston,
USA) for reading the manuscript and providing helpful comments.
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