From the Department of Medicine, Beth Israel
Deaconess Medical Center, Harvard Institutes of Medicine, Harvard
Medical School, Boston, Massachusetts 02115 and the
Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine,
New York, New York 10029
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ABSTRACT |
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Cyclin G, a recent addition to the cyclin family,
was initially identified in screens for new src kinase
family members and soon thereafter by differential screening for
transcriptional targets of the tumor suppressor gene, p53.
We have identified cyclin G as being overexpressed in breast and
prostate cancer cells using differential display polymerase chain
reaction screening. We demonstrate here that cyclin G is overexpressed
in human breast and prostate cancer cells and in cancer cells in
situ from tumor specimens. Cyclin G expression was tightly
regulated throughout the cell cycle in normal breast cells, peaking at
the S and G2/M phases of the cell cycle with lower levels
in G1. The cell cycle-dependent expression was
absent in breast cancer cells. Following DNA damage in normal p53+/+
cells, cyclin G is triggered to cluster in discrete nuclear DNA
replication foci that contain replication-associated proteins such as
proliferating cell nuclear antigen (PCNA). While p53 Human cancer development is a multistage process that results from
the stepwise acquisition of genetic alterations. These alterations may
involve the dysregulation of a variety of normal cellular functions,
leading to the initiation and progression of a tumor. Among normal
cellular functions, regulatory control of the cell cycle plays an
important role in normal cell proliferation, and genetic alterations
that affect cell cycle control have been shown to be associated with
tumor progression (reviewed in Refs. 1-3). Cyclins are prime cell
cycle regulators and control the major check points in cell cycle
transitions of eukaryotic cells (2). In association with a family of
cyclin-dependent protein kinases, cyclins maintain the
orderly progression of cells through the various phases of the cell
cycle. The link between oncogenesis and cyclins has been made with the
aberrant expression of two cyclins (cyclin A and D1) in human tumors
(2, 4-6). Other members of the cyclins (i.e. cyclin B, D3,
and E) have also been shown to have altered expressions in human
tumors, including breast and prostate cancers (7-10).
Cyclin G (Cyc G),1 a recent
addition to the cyclin family, was initially identified in screens for
the src kinase family in rat fibroblasts and soon thereafter
by differential screening for transcriptional targets of the tumor
suppressor gene p53 (11, 12). Cyc G has homology to fission
yeast Cig1, B-type cyclins, and human cyclins A and I (13). Unlike
other cyclin family members, Cyc G lacks a "destruction box" motif
that controls the ubiquitin-dependent degradation but
contains an epidermal growth factor receptor-like autophosphorylation
motif (11, 13). Cyc G is the only known cyclin that is
transcriptionally activated by the p53 tumor suppressor gene, suggesting that it may play a role in p53-mediated cell growth
control (12-16). Furthermore, despite its high homology with other
cyclins, Cyc G has not yet been matched with a
cyclin-dependent kinase binding partner, and its biological
function remains elusive. However, Cyc G forms a complex with B'
regulatory subunits of protein phosphatase 2A following its induction
by p53 (17). In contrast to most p53 target genes such as
p21/Waf1/Cip1/Sdi1, Bax1, IGF-BP, and
Gadd45, Cyc G does not seem to exert a tumor-suppressive role but rather, like other cyclins or proto-oncogenes, plays a
growth-promoting role (15, 18-20). Consistent with this observation, Cyc G overexpression has been observed in human osteosarcoma cells (18,
19).
Initially, we identified the cyc G gene using differential
screening (differential display PCR) (21) between human normal and
tumor breast cells. Here we report the expression pattern, cellular
localization, cell cycle regulation, and Cyc G response to p53
induction in normal and breast cancer cells. We show that aberrant
expression of Cyc G may be closely associated with the tumorigenic
process. Our demonstration that following DNA damage, Cyc G is
triggered to cluster in highly organized nuclear compartments representing DNA replication foci may provide an additional link between p53-mediated growth arrest and cell cycle control. Together, these data suggest that Cyc G may act as an effector in p53-mediated tumor suppression by functional association with replication foci protein(s).
Differential Display PCR and cDNA Library Screening--
The
quality of total RNA was tested by Northern blot analysis before
reverse transcription. Differential display PCR was performed using the
RNAimageTM Kit 1 according to the manufacturer's protocol
(GenHunter). The cDNA products were amplified by the polymerase
chain reaction using H-AP3 5' primer (5'-AAGCTTTGGTCAG-3') and
H-T11G 3' primer. The band of interest was isolated,
PCR-reamplified using the same primers as above, cloned into pGEM-T
vector (Promega), and sequenced by the dideoxynucleotide chain
termination method with Sequenase (U.S. Biochemical Corp.). This
partial-length clone was labeled with [ Cell Lines and Cultures--
Primary human normal mammary
epithelial cells (hNMECs) were established from reduction mammoplasties
obtained through the Cooperative Human Tissue Network and
designated 14N and 15N, as described (23-25). Mouse mammary
epithelial cells (mNMECs) were derived from 129/Sv (p53+/+) and
129/Sv-Trp53 m/Tyj (p53 Cell Cycle Analysis--
hNMECs were synchronized by growth
factor deprivation, lovastatin, or nacodazole block. Synchronization by
growth factor deprivation has been described previously (28). Under
these conditions, cells were arrested at G0 after the
removal of growth factors from the growth media, as determined by
[3H]thymidine incorporation. At time 0, cells were
stimulated to re-enter the cell cycle by the addition of DFCI-1
complete medium with growth factors, and the DNA synthesis rate was
estimated by measuring the incorporation of
[3H]thymidine. Cells cycle synchronization in
G1 was carried out using lovastatin as described (28).
Briefly, hNMECs were grown in D complete medium supplemented with 15 µM lovastatin for 24 h. Cells were induced to enter
the cell cycle by the removal of lovastatin and the addition of 1.5 mM mevalonic acid. Parallel cultures were collected at
several time points to analyze mRNA and protein levels or were
stained with propidium iodide and analyzed for DNA content using
fluorescence-activated cell sorting (FACS) analysis. MCF7 cells were
grown in DMEM, 10% FBS and were synchronized by 20 µM
lovastatin for 33 h, followed by 2.0 mM mevalonic
acid, and were harvested as described above. Nacodazole-induced
G2/M-phase block was done as described (29). Briefly, cells
were grown in medium supplemented with 50 ng/ml nacodazole for 24 h and then nacodazole was removed, cultures were washed, and fresh
growth medium was added. Cells were harvested and analyzed as described above. For FACS analysis, cells were fixed with 80% ice-cold ethanol for 30 min and washed with PBS. Cells were suspended in RNase (10 µg/ml) and incubated at 37 °C for 30 min, washed, and stained for
DNA content with 5 mg/ml propidium iodide. Cells were analyzed on a
FACScan flow cytometer and analyzed with Elite software.
Immunohistoperoxidase Staining--
Immunohistologic studies
were performed on 4-µm frozen sections cut from tissues embedded in
OCT. Tissue blocks were obtained from the Cooperative Human Tissue
Network, eastern division. Immunostaining of sections was performed by
the avidin-biotin peroxidase complex (ABC) method using the Vectastain
Elite ABC kit (Vector Laboratories) as follows. Tissues were treated to
remove endogenous peroxidase activity, blocked with goat serum for 30 min at room temperature, and then incubated in primary anti-Cyc G
antibody (10 µg/ml; UBI) for 2 h. Sections were washed three
times in PBS, 0.1% Tween 20 (PBST) and then incubated with a secondary
anti-rabbit antibody conjugated to horseradish peroxidase for 30 min.
Following PBS washes, the sections were incubated with ABC Elite
reagent for 30 min at room temperature and washed three times in PBS.
The bound horseradish peroxidase complexes were developed using
diaminobenzidine tetrahydrochloride (Sigma Fastdab) according to the
manufacturer's instructions. The sections were counterstained with
hematoxylin, dehydrated, and mounted with glass coverslips.
Immunofluorescence--
Cells were plated onto chamber slides
(Lab-Tek), and when 50% confluent, they were treated with DNA-damaging
agents as described below and then fixed with 100% methanol for 10 min
at Induction of DNA Damage--
Cells were grown to 50%
confluence; MMC was added to cultures at 5 or 10 µg/ml; and cells
were harvested after 6, 12, and 24 h of treatment. Actinomycin D
was added to cultures at a final concentration of 10 or 20 ng/ml and
harvested at 12, 24, and 48 h of treatment. Cells were also
exposed to 6 Gy Western and Northern Blot Analysis--
For Western blot
analysis, samples were adjusted for equal protein and separated on 12%
SDS-polyacrylamide gels under reducing conditions. Gels were
transferred to nitrocellulose membrane, and blots were probed with
antibodies to Cyc G (UBI), Cdc2 (Santa Cruz Biotechnology; catalog no.
054), wild-type p53 (AB-6; Oncogene), and Identification of the Cyc G Gene as a Breast Tumor-specific Gene
through a Differential Display Method--
Using the differential
display PCR technique (21) to survey differentially expressed clones in
human mammary normal and tumor cells, we isolated a cDNA fragment,
15-1 clone, with higher abundance in cancer (T47D and MDAMB435)
compared with normal cells (14N and 15N). After reamplification and
subcloning, 15-1 cDNA fragment was used to confirm the differential
display expression patterns on Northern blot. Sequence analysis and
comparison of 15-1 clone to those within the GenBankTM data
base revealed a 100% sequence match to the 3'-untranslated region of
the human cyclin G cDNA (13, 14). Using this partial clone as a
probe, a normal human mammary cDNA library was screened and a
full-length cDNA was isolated.
Overexpression of Cyc G in Breast and Prostate Cancer
Cells--
We first examined the expression of Cyc G in human mammary
normal and tumor epithelial cell lines. As shown in Fig.
1, all human breast cancer cell lines
examined had higher levels of Cyc G protein expression, regardless of
estrogen receptor status, but a low level of expression was detected in
human normal mammary epithelial cells (14N and 15N). In addition,
higher levels of Cyc G protein were observed in human prostate tumor
cell lines when compared with normal prostate epithelial cells (Fig.
1).
The distribution of Cyc G protein was determined using
immunohistochemistry in a number of human normal and tumor breast
tissue specimens to define the relationship between Cyc G expression and breast cancer development. A total of 40 different cases were analyzed, including 10 normal biopsies, and 30 tumor biopsies (10 in situ carcinomas, seven infiltrating ductal carcinomas, six infiltrating lobular carcinomas, and seven fibroadenomas). Among
the normal biopsies, little or no nuclear or cytoplasmic Cyc G staining
was observed either in the epithelial or stromal cells of the ductal
units (Fig. 2A). However, 22 of the 30 tumor samples showed increased Cyc G expression that was
specific to nuclei of epithelial cells (Fig. 2, C,
E, and G). Positive nuclear staining was observed
in seven of the 10 in situ carcinomas, five of six
infiltrating ductal carcinomas, four of six infiltrating lobular
carcinomas, and six of seven fibroadenomas. With the exception of the
fibroadenomas, stromal cells of the tumor biopsies showed no staining.
Our findings indicate that Cyc G overexpression is a frequent
occurrence in breast cancer.
Cell Cycle-dependent Expression of Cyc G in Normal
Versus Tumor Mammary Epithelial Cells--
To gain further insight
into the possible function(s) of Cyc G, we compared the expression
patterns of Cyc G mRNA and protein throughout the cell cycle in
normal versus tumor cells (Fig.
3, A and B). A
normal human breast epithelial cell strain (15N) and a human mammary
cancer cell line (MCF7) were synchronized by three different methods:
1) lovastatin (cells arrest at G1); 2) growth factor
deprivation (G0; normal cells only); and 3) nacodazole (G2/M). Synchronization of both cell types was monitored by
[3H]thymidine incorporation, histone H4 expression, and
flow cytometry analysis (FACS) (Fig. 3C). Northern and
Western blot analyses of cells harvested at regular intervals revealed
a differential expression pattern of Cyc G during cell cycle
progression in normal versus tumor cells (Fig. 3). In normal
cells (NMECs), Cyc G mRNA and protein levels were low to
undetectable during most of G1 phase. As cells entered into
S phase, a marked induction of Cyc G was observed, which remained high
throughout the S and G2/M phases, correlating with histone
H4 expression (Fig. 3A). A similar pattern of expression was
observed with all three synchronization methods in normal cells.
However, in a human breast cancer line, MCF7, cell
cycle-dependent expression was absent; instead, there was a
consistent high level of expression throughout the cell cycle (Fig.
3B). MCF7 cells were synchronized by lovastatin or nacodazole, both of which gave similar patterns of Cyc G expression (Fig. 3B). Cyc G overexpression in cancer cells may result
from an altered cell cycle regulation.
Differential Induction of Cyc G Expression by p53--
cyc Gis a
transcriptional target gene of p53 that contains two p53-binding
elements in its promotor region (12, 16). High basal levels of Cyc G
expression in tumor cells lacking functional p53 suggest that altered
expression of Cyc G may be independent of p53 dysregulation and may
contribute to the malignant phenotype of cancer cells. To examine the
possibility of an altered response of Cyc G by p53 in some cell types,
we have utilized a tetracycline-regulated p53-inducible system in tumor
cell lines (Saos2-p53 and EJ-p53) that contain nonfunctional p53 and a
high basal level of Cyc G. As shown in Fig.
4A, after tetracycline
removal, p53 mRNA was readily detectable (data not shown) as well
as p21 mRNA, a p53 transcriptional target gene. After removal of
tetracycline in both cell lines, Cyc G levels remained unchanged,
although p21 mRNA was induced dramatically after 12 h,
indicating p53 transcriptional activity. This level of expression
remained consistent and unchanged over time (up to 5 days), suggesting
no specific induction of Cyc G expression by p53 in these cell types.
However, the expression of Cyc G was dramatically induced following
wild-type p53 activation in VhD and Vm10 cells (27), which express low
basal levels of Cyc G (Fig. 4B). The VhD and Vm10 are
immortalized mouse embryo fibroblast cell lines containing a
temperature-sensitive p53 mutant (Val to Ala mutation at codon 135, tsp53), which express a nonfunctional p53 at 37-39 °C and fully
functional p53 at 32 °C. The VhD cells undergo reversible
G1 arrest in a p53-dependent manner at
32 °C, while the Vm10 cell line, which constitutively expresses
tsp53 and the c-myc oncogene undergoes apoptosis at 32 °C
(27). The expression of p21 in these two tsp53 cell lines was low
before the temperature shift and increased with similar kinetics to Cyc G induction. Altogether, these data demonstrate that the activation of
Cyc G expression by p53 induction or activation is universal for both
growth arrest and apoptotic pathways but only observed in cells
containing low basal expression of Cyc G.
Localization of Cyc G to Nuclear Replication Foci in Response to
DNA Damage--
In order to determine regulation and localization of
Cyc G in response to DNA damage, we first examined the expression and localization of Cyc G in mNMECs isolated from p53-deficient mice after
treatment with DNA damaging agents. Cells from the mammary glands of
p53 null mice as well as cells from wild type mice were treated with
actinomycin D (not shown) or MMC. Northern blot analysis showed that
Cyc G mRNA expression was induced after treatment with actinomycin
D in wild-type cells, while in p53 null cells, Cyc G mRNA was
hardly detectable, and no induction was observed after DNA damage
treatment (Fig. 5A,
top). We also examined the cells for distribution of Cyc G
protein before and after DNA damage with MMC using immunofluorescence
staining (Fig. 5A, bottom). In wild-type cells,
the staining with anti-Cyc G antibodies revealed an amplification of
distinct punctate patterns in nuclei following DNA damage treatment,
while untreated cells had a lower number of punctate nuclear signals.
These MMC-treated wild-type cells also showed strong recruitment of p53
to the nuclei. However, p53 null cells displayed a very faint Cyc G
nuclear staining pattern, and there was no increase of signals after
DNA damage (Fig. 5A).
We next compared the distribution of Cyc G before and after DNA damage
in human normal and tumor mammary epithelial cells (Fig.
5B). When 15N cells (hNMEC) were methanol-fixed and stained for Cyc G, a distinct but low level nuclear signal was detected (Fig.
5B), similar to that seen in p53+/+ mNMEC (Fig.
5A). The staining correlated well with the Western blot
analysis, which showed low levels of Cyc G protein in NMECs compared
with tumor cell lines (Fig. 1). After exposure to MMC, a dramatic
amplification of Cyc G signal was observed, which was localized to the
nucleus in a discrete staining pattern, consisting of 6-10 brightly
labeled clusters per cell reminiscent of DNA replication foci (Fig.
5B). In human mammary cancer cells, the patterns of Cyc G
localization were not as distinct as those seen in normal cells,
although the staining was still present in the nucleus (Fig.
5B). We used two tumor mammary cancer cell lines: MCF7,
containing wild-type p53, and T47D, containing a
nonfunctional mutated p53 gene (30). Both lines have higher
basal expression levels of Cyc G as compared with hNMEC (Fig.
5B). MCF7 cells displayed amplified nuclear staining signals
in response to DNA damage, but T47D cells did not show any induction of
Cyc G protein by DNA damage (Fig. 5B). The distribution of
staining for both cell types also appeared predominantly nuclear.
The localization observed after DNA damage in normal cells suggests
that Cyc G may be recruited to areas of DNA replication and repair,
delineated as DNA replication foci, where proteins such as PCNA and
several cyclins including cyclin A and D have been shown to colocalize
(31). Given the pattern of Cyc G localization in the nucleus and the
potential involvement of Cyc G in DNA replication and repair, we
explored the possibility of an association between Cyc G and PCNA,
which plays an essential role in nucleic acid metabolism as a component
of the replication and repair machinery. Co-localization between Cyc G
and PCNA proteins was tested on MMC-treated (10 µg/ml for 12 h)
15N cells using two-color immunostaining (Fig.
6). Detergent extraction of the cells
followed by methanol fixation removes detergent-soluble PCNA, and
enables the clear visualization of detergent-insoluble PCNA, the form
that is tightly complexed to DNA in replication foci (31-34). A
colocalization of PCNA and Cyc G was observed in defined foci in the
nuclei of DNA-damaged cells. To assess whether a PCNA/Cyc G
co-localization pattern was indicative of an association between the
two proteins, immunoprecipitation analyses were also carried out.
However, we failed to detect a co-immunoprecipitation complex between
these two proteins, raising the possibility that Cyc G may function at
the site of DNA replication but does not directly interact with
PCNA.
Accumulating evidence suggests that altered expression of cyclins,
including cyclins A, D1, and E, may contribute to the malignant phenotype of human cancers such as mammary and prostate cancers (5-10). Ongoing efforts in the discovery of candidate genes whose function or expression is altered during the course of breast carcinogenesis led to the identification of Cyc G whose expression was
up-regulated in breast and prostate cancer cells both in
vitro and in vivo. In an attempt to understand the
potential role of Cyc G in the process of tumorigenesis, we have
studied its expression pattern, cell cycle regulation, response to DNA
damage, and subcellular distribution in normal and cancerous cells.
To assess the in vivo relevance of Cyc G overexpression in
breast and prostate cancer cell lines, we examined its expression pattern in clinical breast cancer specimens as well as normal breast
tissues by immunohistochemistry. Consistent with the high levels of
expression observed in cell lines, a significant Cyc G overexpression
occurs in the majority of in situ and invasive ductal and
lobular carcinomas examined. Cyc G staining is restricted to the nuclei
in the malignant tumor biopsies, whereas staining in normal ductal
units is barely detectable. This observation is similar to cyclin D1
overexpression in the ductal type of both noninvasive and invasive
breast cancers when compared with more benign breast diseases, which
express lower levels of cyclin D1. In about 15% of these cases, the
cyclin D1 overexpression appears to result from gene amplification
(10). In addition, we found that six of seven fibroadenomas examined
showed positive staining for Cyc G, suggesting that aberrant
overexpression of Cyc G may occur during the earlier stages of
tumorigenesis. That Cyc G was overexpressed predominantly in
transformed cells in vitro and in vivo suggests
that Cyc G is deregulated during oncogenesis and has potential
diagnostic/prognostic value in cancer development. Others have reported
the overexpression of Cyc G in osteosarcoma cells (19, 20) and reported
that antisense Cyc G expression inhibited growth of human osteogenic
sarcoma cells in vitro and in a nude mouse in
vivo tumor model and resulted in a significant increase in the
incidence of apoptosis (19, 35). In addition, ectopic overexpression of
Cyc G in human RKO colon carcinoma cell line accelerated cell growth,
and the transfection of normal human fibroblasts with Cyc G increased
their proliferative lifespan (15). These data, as well as our findings,
suggest that Cyc G may possess or correlate with oncogenic potential.
However, Cyc G has also been identified as a transcriptional target
gene for the tumor suppressor gene p53, and its promoter
sequence contains functional p53 binding sites that efficiently respond
to p53 (12, 16). In contrast to other p53-regulated genes, Cyc G
appears to behave much like other cyclins or protooncogenes. Although several possible roles of Cyc G have been speculated in a p53-mediated tumor suppression, the function of Cyc G remains elusive, and its
classification as a cyclin rests solely on sequence similarities with
other members of the cyclin family.
It has been demonstrated that Cyc G is constantly expressed throughout
the cell cycle without any specific pattern in HeLa cells (11, 12, 14).
In other studies in which they examined Cyc G expression during the
cell cycle in tumor cell lines such as murine B cell lines and NRK-49F
cells, there was no change in Cyc G expression levels throughout the
cell cycle (11, 13, 14). Consistent with the previous data, we have
found that Cyc G is constitutively expressed in two breast cancer cell
lines in all phases of the cell cycle. However, like other members of the cyclin family, Cyc G expression is gradually increased with cell
cycle progression, peaking in late S and G2/M phases in
human normal mammary epithelial cells. Thus, constitutive Cyc G
expression during the cell cycle progression may contribute to the
dysfunction of cell cycle control in transformed cells, which is
tightly regulated in normal cells.
We have observed that expression and induction of Cyc G parallels that
of p53 in human NMECs after DNA damage. Although cyc G is a
transcriptional target gene of p53, its overexpression in the majority
of breast and prostate cancer cells does not simply reflect the p53
status in those cells; i.e. whether there exists wild type
p53 in cells (MCF7) or a mutated form (T47D), Cyc G is expressed at
higher than normal levels. This may be due, in part, to other
mechanisms that regulate Cyc G expression independent of p53. It is
clear that ectopic wild-type p53 expression in p53 null tumor cells can
transactivate expression of p53 target genes by severalfold (36, 37).
Our studies utilizing a tetracycline-regulated p53 inducible system in
two p53-null tumor cell lines (Saos2, EJ) demonstrate that Cyc G levels
remain unchanged following p53 overexpression, although other
p53-inducible genes such as p21 and mdm2 were
dramatically induced in a short time. Despite persistent ectopic p53
expression and transcriptional activity, the level of Cyc G expression
remained consistent and unchanged for up to 5 days in both EJ and Saos2
cells. However, the expression of Cyc G was dramatically induced
following wild-type p53 activation in VhD and Vm10 cells, which express
low basal levels of Cyc G (Fig. 4B). The VhD and Vm10 cells
are immortalized mouse embryo fibroblast cell lines that contain a
temperature-sensitive p53 mutant (tsp53), but they do not appear to
assume malignant phenotypes (27, 38). Upon a shift to the permissive
temperature and p53 activation, VhD cells become arrested in
G1, whereas Vm10 cells undergo apoptosis (39). These data
indicate that when the basal expression levels are high, Cyc G becomes
unresponsive to p53 transcriptional activation. They also demonstrate
that, at least in artificial systems where p53 is ectopically
expressed, the expression of Cyc G is increased irrespective of
subsequent cell fate, whether it is growth arrest or apoptosis.
Nonetheless, the Cyc G response to p53 is only seen in cells containing
the low basal expression of Cyc G (e.g. normal cells) or in
nonmalignant cells. Although the mechanisms responsible for this
differential response of Cyc G induction by p53 remain to be
elucidated, one possibility is that Cyc G mRNA might be stabilized
in tumor cells and that high basal levels of Cyc G expression may act
in a negative feedback loop, suppressing p53-mediated Cyc G
transcription. Another possibility is that in tumor cells, Cyc G is
being transcribed independent of p53 transcriptional activity at a
maximal rate with no further increase in transcription upon p53
induction/activation. Therefore, the constitutive expression of Cyc G
may account for dysregulation of the normal cell cycle in cancer cells,
hence affecting their response to various stimuli such as DNA damage.
It is known that in the mammalian nucleus, DNA is replicated in
discrete nuclear foci and that PCNA, a replication-associated protein,
specifically localizes at these replication foci. Previously, a direct
link between cell cycle regulation and DNA replication was established
when it was shown that Cyc A and cyclin-dependent kinase 2, a complex needed for entry into S phase, specifically localize at
nuclear replication foci (39). Given that Cyc G is dramatically
recruited to replication foci following DNA damage in normal cells, it
is likely that Cyc G acts as an effector in p53-mediated events by
functional association with one or more of the replication foci
proteins. Our attempts at elucidating one such association, namely, Cyc
G and PCNA, by co-immunoprecipitation did not reveal any physical
association between these two proteins. Nonetheless, the subcellular
colocalization of Cyc G at DNA replication foci suggests a role for Cyc
G in regulating DNA replication. The demonstration of colocalization by
itself does not prove Cyc G's involvement in DNA replication, but it
is consistent with the speculation that in association with replication
protein(s), Cyc G may act as an important regulator of cell
proliferation and reponse to DNA damage in normal cells. Recent
experiments have demonstrated that Cyc G-immunoprecipitated complexes
collected from Cyc G transfectants displayed an appreciable level of
cyclin-dependent kinase activity and contained the
retinoblastoma protein (15), thus suggesting potential involvement of
Cyc G during the cell cycle progression. Further studies are needed to
identify Cyc G-associated proteins and their relevance to its potential
growth arrest function.
Cultured cells from Ataxia-Telangiectasia patients (A-T cells) are
unable to arrest at the G1-S boundary following DNA damage such as irradiation. A recent report suggested that Cyc G may contribute to G2/M arrest of cells in response to DNA
damage (40). From our initial efforts to define whether Cyc G responds
similarly to p53 and its target gene p21 following DNA
damage in A-T cells, five out of six AT cell lines revealed an aberrant
Cyc G response following DNA damage treatment with actinomycin D (10 ng/ml) when compared with normal dermal fibroblasts, while these five
A-T cell lines as well as the normal dermal fibroblasts showed an increase of p53 protein and p21
expression.2 Thus, altered
response of Cyc G by DNA damage in A-T cells may contribute to the DNA
repair defect of these A-T cells.
The present study demonstrates that Cyc G is overexpressed in breast
cancer tissues and cell lines, independent of p53 status. Although the
Cyc G promoter contains two p53 response elements, the transcriptional
regulation of Cyc G by p53 is highly dependent on the cellular context
in which p53 is activated. Our data demonstrate that Cyc G is expressed
with periodicity throughout the cell cycle of normal cells, but not in
tumor cell lines, and that localization of Cyc G at replication foci is
quite distinct and may provide a clue for involvement in DNA
replication. Finally, overexpression of Cyc G in human cancers such as
breast and prostate may prove to be an important prognostic marker for
tumor initiation and/or progression. Future studies should be directed
at testing the diagnostic value of Cyc G on a larger scale to firmly
establish a correlation between the degree of Cyc G expression and
breast or prostate cancer progression. It is also conceivable that new therapeutic approaches directed specifically at down-regulating Cyc G
in breast and prostate cancers might cause a reduction of tumorigenic potential.
/
cells
displayed a faint cyclin G nuclear staining pattern, there was no
increased expression and no change in distribution of the staining
pattern after DNA damage. The specific subcellular localization of
cyclin G at DNA replication foci provides an additional link between
p53-mediated growth arrest and cell cycle regulation and suggests that
cyclin G may act as an effector of p53-mediated events by functional
association with replication foci protein(s).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using
the random primed synthesis method and used as a probe to obtain the
full-length clone by screening a cDNA library prepared from normal
mammary epithelial cells (22).
/
) mice obtained from Jackson Laboratories.
These cells were grown in DFCI-1 medium (D Complete) as described (24)
and were used at early to mid passage, i.e. 5-10 population
doublings. Human breast cancer cells (MCF7, T47D, Hs578t, MDAMB435, and
MDAMB436) were obtained from ATCC. Growth media for these mammary
cancer cells was DMEM, 10% fetal bovine serum (FBS). Primary human
normal prostate cells were derived from normal adjacent tumor tissue
biopsies received from the Cooperative Human Tissue Network and
designated NPrEC-1 and -2. These cultures were grown in Clonetics
Prostate Epithelial Media. The human prostate carcinoma cell lines PC3, LNCaP, and DU145 were purchased from the ATCC and maintained in DMEM,
10% FBS. ND1 was kindly provided by Dr. P. Narayan. EJ-p53, EJ-CAT
cells, and Saos 2-p53 cells were cultured in the presence or absence of
tetracycline (1 µg/ml) in DMEM, 10% FBS as described previously
(26). To induce the expression of p53, cells were washed three times
with PBS, and fresh medium without tetracycline was added. The murine
cell lines Vm10, VhD, and 10.1 were a gift from Xiangwei Wu (Mount
Sinai School of Medicine) and were cultured in DMEM, 10% FBS plus
hygromycin (50 µg/ml) as described (27).
20 °C, followed by permeabilization with 0.1% Triton X-100 for
5 min. Staining was carried out as follows. Cells were blocked with
PBST containing 10% heat-inactivated goat serum, 3% nonfat dry milk for 30 min at room temperature. Primary antibodies (Cyc G; Santa Cruz
Biotechnology Inc. (Santa Cruz, CA) catalog no. SC-320) or nonspecific
rabbit IgG (10 µg/ml) was diluted with 0.1% nonfat milk/PBST. Cells
were incubated for 1.5 h, rinsed in PBST, and then stained with a
rhodamine-conjugated secondary antibody for 1 h. Cells were then
observed with a Zeiss Axioscope I photomicroscope equipped with
epifluorescence. Cyc G antibodies (UBI and Santa Cruz Biotechnology)
and anti-PCNA (clone PC10; Oncogene) were used for dual labeling.
Double immunofluorescence labeling was carried out with two different
secondary antibodies specific to each primary antibody, and double
exposures were taken. Co-localization between Cyc G (red, rhodamine)
and PCNA (green, fluorescein isothiocyanate) proteins was tested on
mitomycin C (MMC; 10 µg/ml)-treated hNMEC (15N) cells using two-color
immunostaining. Detergent extraction of the cells followed by methanol
fixation removed detergent-soluble PCNA and enabled the clear
visualization of detergent-insoluble PCNA, the form that is tightly
complexed to DNA in replication foci (23).
-irradiation in a Mark 137I cesium
source irradiator and recovered for 12 h. Cells were collected in
reducing SDS-polyacrylamide gel electrophoresis loading buffer and
boiled for 5 min for Western blots or collected in guanidium
isothiocyanate buffer for RNA extraction.
-actin (clone AC-15;
Sigma). Bands were detected using the ECL chemilluminescence detection
method (Amersham Pharmacia Biotech) and exposed to x-ray film. For
Northern blot analysis, total RNA was extracted, denatured, and
electrophoresed through a 1% agarose-formaldehyde gel (20 µg of
total RNA/lane). The gel was transferred to nylon membrane (Bio-Rad)
and hybridized as follows. Human Cyc G, p21, p53, histone H4, and 36B4
probes were 32P-labeled by using random primed DNA labeling
techniques. Blots were exposed on x-ray film after washing. In all
cases, films were scanned (HP ScanJet IIcs) and analyzed using Adobe
Photoshop software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of cyclin G in human mammary
and prostate carcinoma cell lines. Western blot analysis was used
to determine the level of Cyc G protein in normal cells derived from
hNMECs (cell strains 14N (primary cultures) and 15N (passage 7 cultures)) and in several mammary epithelial tumor cell lines MCF7,
T47D, Hs578t, MDAMB435, and MDAMB436. Cyc G protein expression was also
measured in normal primary (NPrEC-1 and -2) and tumor prostate cell
lines LNCaP, DU145, PC3, and ND1. Aliquots of cell lysates derived from
the indicated cell lines were loaded in each lane and then separated by
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membrane and then immunoblotted with
antibodies Cyc G and -actin, respectively. Immunoblotting for
-actin was used for equal loading of protein samples.
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Fig. 2.
Immunohistological detection of Cyc G
expression in human normal and cancerous breast tissue specimens.
Paraffin sections were stained using Cyc G antibodies. Immunoperoxidase
staining of normal (A), in situ carcinoma
(C), infiltrating ductal carcinomas (E), and
fibroadenoma (G) is shown. Note that higher magnifications
demonstrate strong nuclear staining of epithelial cells
(insets of C, E, and G).
Control IgG staining of subadjacent tissues showed very low background
signal (B, D, F, and H).
Bar, 100 µM (inset bar,
15 µM).
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Fig. 3.
Cell cycle expression of Cyc G in normal
(15N) and tumor (MCF7) mammary epithelial cells. 15N and MCF7
cells were synchronized by lovastatin (at G1) and
nacodazole (at G2/M). A, cell
cycle-dependent expression of Cyc G in 15N cells. Normal
cells (15N) were also synchronized by growth factor starvation (at
G0). Cell cycle progression was monitored by the level of
histone H4 mRNA (G1/S marker), Cdc2 protein
(G2/M marker), FACS analysis (shown as a table),
and [3H]thymidine incorporation (not shown). Northern and
Western blot analysis of Cyc G showed a marked induction shortly after
entry into S phase in human NMECs synchronized by three methods.
B, Cyc G expression in synchronized MCF7 cells during cell
cycle progression. MCF7 cells were synchronized by lovastatin or
nocodazole treatment. Total RNA and protein extracts were isolated from
synchronized cells and analyzed for Cyc G expression throughout the
cell cycle. Unlike the case with normal 15N cells, Northern and Western
blot analysis of MCF7 cells revealed a constant and high level of Cyc G
mRNA and protein expression, showing no apparent regulation through
the cell cycle. C, cell cycle analysis of synchronized cells
by FACS. Populations of cells in different phases of the cell cycle are
shown.
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Fig. 4.
p53-dependent expression of Cyc G
in different cell types. Total RNA was isolated from the indicated
cells following p53 induction by tetracycline-regulatable systems
(A, EJ-p53 and Saos2-p53) or temperature shift (from 38 to
32 °C) (B, Vm10, Vhd, and 10.1), and Northern blots were
performed to determine the induction of Cyc G, p21, and 36B4. Each lane
contains 20 µg of total RNA.
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Fig. 5.
Cellular localization of Cyc G and its
response to DNA damage. A, top,
p53-dependent Cyc G induction following mitomycin C
treatment (10 µg/ml) in p53+/+ versus p53 /
mouse
normal mammary epithelial cells (mNMECs). p53
/
or +/+ mNMECs were
treated with/without DNA damage agent, MMC, for 24 h, and total
RNA was isolated for Northern blot analysis. The Northern blot was
consecutively hybridized with Cyc G, p21, and 36B4 probes. 36B4 was
used a loading control. Bottom, cellular localization of Cyc
G with/without MMC in p53+/+ and p53
/
mNMECs. Cells were incubated
MMC (10 µg/ml) and fixed in methanol and immunostained for Cyc G and
p53. No increase in Cyc G staining was observed in p53 null cells.
Bar, 15 µM. B, specific
localization of Cyc G by DNA damage in human mammary normal
versus cancer cells. 15N, MCF7, and T47D cells were
incubated for 12 h with/without MMC (10 µg/ml). The cultures
were fixed and stained for Cyc G antibodies for cellular localization.
Notice that the staining was restricted to the nucleus and was in a
characteristic pattern, consisting of 5-9 distinct clusters in each
cell in 15N cells. In two mammary tumor cell lines, MCF7 and T47D, Cyc
G staining also appeared predominantly nuclear, although the discrete
clusters of labeling were not as pronounced, as was the case in normal
cells (15N). Instead, the punctate staining in the tumor cells appeared
to be distributed throughout the nucleus with little overt spatial
organization. No major increase of Cyc G labeling was observed
following DNA damage. Bar, 10 µM.
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Fig. 6.
Colocalization of Cyc G and PCNA in a
discrete nuclear pattern in DNA damage treated hNMECs. 15N cells
were treated with MMC (10 µg/ml) for 12 h, fixed with methanol,
treated with Triton X-100, and stained with both anti-Cyc G and PCNA
antibodies. PCNA (green) is strictly nuclear and has a
punctate pattern; Cyc G (red) appears nuclear in these cells
as well. There was extensive colocalization of Cyc G (red)
and replicating PCNA (green) as indicated by
yellow signals in the composite picture.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. X. Wu for the tsp-p53 cell lines and Dr. P. Narayan for the ND1 cell line. We thank K. Claffey for useful suggestions and D. Campbell and M. Tang for technical help.
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FOOTNOTES |
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* This work was supported by National Institutes of Health grants and a grant from the Massachusetts Department of Public Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
¶ A Howard Hughes Medical Institute Medical Student Research Training Fellow.
** To whom correspondence should be addressed: Harvard Institutes of Medicine, Rm. 921, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-667-8563; Fax: 617-667-0980; E-mail: slee2{at}bidmc.harvard.edu.
2 C. L. Reimer, P. Arizti, S. K. Kurdistani, J. R. Garreau, and S. W. Lee, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: Cyc G, cyclin G; NMEC, normal mammary epithelial cell; hNMEC, human normal mammary epithelial cell; mNMEC, mouse normal mammary epithelial cell; FACS, fluorescence-activated cell sorter; MMC, mitomycin C; PCR, polymerase chain reaction; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; ABC, avidin-biotin peroxidase complex; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen.
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