Transfection of an Inducible p16/CDKN2A Construct Mediates Reversible Growth Inhibition and G1 Arrest in the AtT20 Pituitary Tumor Cell Line
Simon J. Frost,
David J. Simpson,
Richard N. Clayton and
William E. Farrell
Centre for Cell and Molecular Medicine School of Postgraduate
Medicine Keele University North Staffordshire Hospital
Stoke-on-Trent, ST4 7QB. United Kingdom
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ABSTRACT
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Recent studies have shown that methylation of the
CpG island within the p16/CDKN2A gene is associated with an absence of
p16 protein in human pituitary tumors. However, the effect of
restoration of p16 protein expression in this tumor type has not been
investigated.
In the absence of an available human pituitary cell line we first
assessed the suitability of the mouse corticotroph cell line AtT20 as a
model system. Initial experiments showed that the p16/CDKN2A gene was
not expressed, whereas a transcript for RB1 was detected as
assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be
homozygously deleted. The absence of p16/CDKN2A and presence of
RB1, the downstream effector of p16-mediated cell cycle
arrest confirmed the suitability of the AtT20 cell line as a
model system. Stable transfectants were generated in which
p16/CDKN2A is regulated by an inducible promoter. The regulatory
effects of p16/CDKN2A expression on cell proliferation were assessed
and complemented by fluorescence-activated cell sorting
(FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A
resulted in a profound inhibition of cell growth and
G1 arrest (8082%). Western blot analysis
showed concomitant expression of p16 protein in arrested cells and a
shift in the phosphorylation status of pRB toward its
hypophosphorylated form. To further confirm that expression of
p16/CDKN2A mimicked its in vivo role, reversibility was
assessed using alternate cycles in the presence and absence of inducer
(isopropyl-1-thio-ß-D-galactopyranoside).
Over three cycles the absence of induced expression of p16/CDKN2A
resulted in release from G1 arrest.
These results show that, in a pituitary cell line model, restoration of
p16 expression is indeed sufficient to arrest cells in
G1 and inhibit cell proliferation and is
reversible. Thus restoration of p16 expression through novel
strategies, including gene therapy or demethylating agents, may offer
successful therapeutic intervention in human forms of this disease.
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INTRODUCTION
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Pituitary tumors are common benign neoplasms of the
adenohypophysis representing approximately 10% of all adult
intracranial neoplasms (1). However, a proportion of adenomas may
display aggressive behavior and extend into the adjacent bone and sinus
tissue, while a very small proportion have the potential to become
malignant and may metastasize beyond the central nervous system (1, 2).
Studies of X chromosome inactivation have shown that pituitary tumors
are predominantly monoclonal in origin, suggesting that they arise from
clonal expansion of a single genetically altered cell (3). Initiation
and progression of endocrine tumors are thought to arise from the
accumulation of both genetic (4, 5, 6, 7) and, more recently, epigenetic
aberrations (8, 9) .
Regulation of cell cycle progression from G1 to S phase is
tightly controlled by three groups of proteins, cyclins,
cyclin-dependent kinases (CDKs), and CDK inhibitors. Cyclin D1/CDK4
complexes catalyze the first cascade of pRB phosphorylation in late
G1 phase, resulting in release of the E2F family of
transcription factors and allowing expression of genes critical for S
phase progression (10). Cyclin D1 and p16 are the positive and negative
regulators of CDK4, respectively, and thus regulate the phosphorylation
status of the RB protein (11, 12). While p16 protein inhibits
the activity of CDK4 (12), the D type cyclins (D1, D2, and D3) activate
CDK4 (13). Thus, p16 can specifically associate with CDK4 and disrupt
the formation of active kinase complexes (14) and prevent transition of
cells from G1 to S phase of the cell cycle (15). The p16
locus (9p21) has been shown to be frequently disrupted by hemi- or
homozygous deletion in a variety of human tumor cell lines (16, 17, 18)
and, to a lesser extent, in primary tumors (reviewed in Ref. 19).
However, analysis of human pituitary tumors showed that hemizygous
deletions flank but exclude the p16 gene (20). Although p16 has been
shown to be a critical cell cycle regulator in many cell types, it is
not expressed in normal tissue from colonic mucosa (21), breast (22),
or in normal brain tissue from patients with gliomas (23). This
suggests that, at least in certain tissues, other mechanisms
(independent of p16) may mediate the G1-S transition. In
addition, transfection of cell lines from a variety of tumor types,
including esophagus, lung, liver, ovary, and mesothelioma, with p16
constructs has been shown to have a variable effect on cell
proliferation (24).
Studies in numerous human tumor types, including pituitary, have shown
that methylation of the CpG island that extends into exons 1 and 2 of
the p16/CDKN2A gene is associated with gene silencing (9, 21, 25, 26).
A recent report showed (27) that the majority of pituitary adenomas
studied failed to express detectable p16 protein as assessed by Western
blot analysis. Subsequent studies (8, 9) showed that methylation of the
CpG island within p16/CDKN2A was a frequent event in this tumor type
and was significantly associated with loss of p16 protein expression.
While a clear relationship between methylation of the p16/CDKN2A gene
and loss of protein expression has been demonstrated for several tumor
types (21, 25, 26), including the pituitary (8, 9), the effects of
restoration of p16 protein expression in the pituitary has not been
demonstrated.
The methylation status of the p16/CDKN2A gene (and of other TSGs) is
frequently assigned by analysis of the CpG island within the coding
regions of the gene (8, 9, 21, 28) and thus, by association, reflects
the methylation status of the upstream promoter region. Methylation of
the coding region is presumed to represent an epiphenomenon of
methylation-induced promoter silencing and is thought to reflect a
consequence rather than a mechanism of gene silencing (21). Although a
clear link between CpG island promoter methylation and gene silencing
has been established through in vitro methylation and
transfection studies (29, 30, 31, 32, 33) where studied, methylation within coding
region CpG islands is not associated with decreased expression (21, 33).
In the absence of a suitable human pituitary cell line and due to the
difficulty of propagating primary tumors in vitro, we first
assessed the suitability of the mouse pituitary corticotroph cell line
AtT20 as a model system. Analysis of p16/CDKN2A and RB1
transcript expression in the AtT20 cell line showed that pRB is
expressed and p16/CDKN2A is lost, and the mechanism responsible for
p16/CDKN2A loss was found to be homozygous deletion. Therefore, this
cell line represents a suitable model for assessing the role of
p16/CDKN2A on pituitary tumor cell growth.
In this study we describe the generation of a stably transfected AtT20
cell line in which an inducible promoter regulates ectopic expression
of the p16/CDKN2A gene. This model system has allowed us to determine
the effect of restoring p16/CDKN2A expression in a pituitary tumor cell
line lacking this gene.
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RESULTS
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p16/CDKN2A and RB1 Status of the AtT20 Cell
Line
Since no suitable human pituitary cell line is available, the
mouse pituitary tumor cell line AtT20 was first assessed with regard to
its p16/CDKN2A and RB1 status before the studies described
below. After cDNA synthesis, expression of the p16/CDKN2A transcript
was assessed by PCR amplification of cDNA from AtT20 cell line and
normal mouse pituitary. Using mouse-specific PCR primers, the
p16/CDKN2A transcript was undetectable in the AtT20 cell line (Fig. 1a
). Fig 1a
also shows p16/CDKN2A
transcript expression in normal mouse pituitary as assessed by RT-PCR
analysis. Further analysis of the p16/CDKN2A gene in the AtT20 cell
line (with primers specific for genomic DNA) showed the p16/CDKN2A gene
to be homozygously deleted in this cell line (data not shown). Since
pRB is a critical downstream effector of p16/CDKN2A-mediated growth
inhibition, we analyzed the transcript expression of RB1 in
AtT20 cells and normal mouse pituitary by RT-PCR and confirmed
expression in this cell line and normal mouse pituitary (Fig. 1b
).
Thus, the absence and presence of p16/CDKN2A and RB1,
respectively, in the AtT20 cell line allowed us to assess the
contribution of p16/CDKN2A to growth control in a model pituitary
system.

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Figure 1. RT-PCR Analysis of Endogenous p16/CDKN2A (a), RB1
(b), and Exogenous p16/CDKN2A (c) Transcript in the Stably Transfected
AtT20 Cell Line Clone Designated AtT20/p16+ and in Normal Mouse
Pituitary
Endogenous p16/CDKN2A is not expressed in AtT20 cells (a) whereas a
transcript for RB1 was detected (b). Analysis of normal
mouse pituitary shows expression of both endogenous p16/CDKN2A and
RB1 transcripts in this tissue. Panel c shows p16/CDKN2A
transcript expression in the presence of IPTG, but not in the absence
of the inducer (-IPTG). The housekeeping gene Porphobilinogen
Deaminase (PBGD) was coamplified in a multiplex PCR reaction to control
for cDNA synthesis and RNA integrity. The lane marked M represents mol
wt markers, with the sizes shown on the left of the
figure.
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Effect of p16/CDKN2A Reexpression on AtT20 Cell Line Growth
in Vitro
To generate a stably transfected pituitary tumor cell line, in
which p16/CDKN2A expression could be induced, we used an
isopropyl-1-thio-ß-D-galactopyranoside (IPTG)-regulated
expression system. Using the lac-switch technology, p16/CDKN2A
expression is repressed by interaction of the lac repressor protein
with the operon that drives p16/CDKN2A expression. Expression of
p16/CDKN2A is induced by addition of IPTG to the growth medium. After
stable transfection, clones were screened for expression of the lac
repressor by RT-PCR. Clones that expressed high levels of lac
repressor, as assessed by semiquantative RT-PCR (data not shown), were
selected for further analysis. A representative example of IPTG-induced
ectopic p16/CDKN2A transcript expression (as assessed by RT-PCR) is
shown (Fig. 1c
). In the 4 of 10 clones originally isolated that did not
show a reduction in colony-forming efficiency (see Materials and
Methods), we failed to detect expression of a p16/CDKN2A
transcript as assessed by RT-PCR analysis. After induction with IPTG,
the growth response of a clone designated AtT20/p16+ to IPTG is shown
in Fig. 2
. Varying the dose of IPTG from
0.25 mM resulted in a dose-dependent inhibition of colony
forming efficiency. At the highest dose employed (5 mM),
there is an 85% reduction in colony forming efficiency in comparison
to transfected cells grown in the absence of IPTG. The reduction in
colony forming efficiency was not due to cytotoxicity as assessed by
trypan blue exclusion. The possibility of IPTG alone reducing colony
forming efficiency was excluded by control experiments in untransfected
cells in the absence and presence of 5 mM IPTG. No
reduction in colony forming efficiency was evident.

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Figure 2. Dose Response of a Single Stably Transfected Clone
AtT20/p16+ to IPTG as Shown by Inhibition of Colony Forming Efficiency
Cells were treated with 0.25 mM IPTG for 72 h and
the number of colonies counted. The number of colonies formed in the
untreated controls was expressed as 100% colony forming efficiency.
The results are expressed as the mean ± SD of three
independent experiments, with each experiment performed in triplicate.
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Since the colony forming efficiency studies showed a dose-dependent
effect of enforced p16 expression, we assessed the effects of induced
p16 expression on cell proliferation over a 14-day growth curve. Stably
transfected cells, harboring either the inducible p16 construct or an
empty vector, were grown in the presence or absence of the inducing
agent IPTG. Figure 3
shows that for the
clone AtT20/p16+ growth in the presence of 5 mM IPTG (Fig. 3a
) results in a near-complete cessation in cell proliferation that was
maintained throughout the experiment. The growth rate of cells in 0.2
mM IPTG (Fig. 3b
) showed an intermediate reduction in cell
proliferation, consistent with the partial decrease in growth evident
in the colony forming efficiency studies (Fig. 2
). Figure 3c
shows the
growth curves of cells incubated in the absence of IPTG, which were
stably transfected with a construct either harboring p16 (AtT20/p16+)
or with the empty expression vector [AtT20/p16 (-)]. Both clones
showed similar growth patterns throughout the experiment. These results
indicate that the p16 construct in the absence of inducer did not
inhibit cell proliferation (Fig. 3c
).

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Figure 3. Cell Growth Inhibition Studies of Stably
Transfected AtT20 in the Presence of 5 mM (a), 0.2
mM IPTG (b), and Absence of IPTG (c)
Open circles represent clone AtT20/p16+ shown to
harbor an inducible p16 construct (see Figs. 1 and 2 ). Solid
triangles represent clone AtT20/p16 (-) harboring an
"empty" inducible expression vector. At each time point triplicate
dishes were sacrificed and the total number of cells was determined
(ordinate) and expressed as viable cell counts. The
values represent the mean, and the bars represent the SD
from triplicate determinations and are representative of three
independent experiments. Irrespective of treatment the percentage of
nonviable cells (as assessed by trypan blue exclusion) did not differ
significantly throughout the experiment and never exceeded 10% of
total cell counts. At each time point, culture media was replaced
together with the appropriate concentration of IPTG. For cultures
incubated in the absence of IPTG, cells were challenged with vehicle
alone.
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Induction of p16 Expression Causes G1
Arrest in Stably Transfected AtT20 Cell Line
To determine the mechanism responsible for the reduction in cell
proliferation, AtT20 cells stably transfected with the lac repressor
and inducible p16 construct were first subject to
fluorescence-activated cell sorting (FACS) analysis. Cells were
harvested 3 days after induction with IPTG (5 mM) to
determine their cell cycle profile after enforced reexpression of
p16/CDKN2A. Figure 4
shows that, after
induction with 5 mM IPTG, expression of p16/CDKN2A
increases the number of cells in the G1 phase of the cell
cycle in the clone AtT20/p16+ from 58% to 82%. Table 1
summarizes the results of FACS analysis
for a total of four independently isolated clones and shows, in each
case, a similar proportion of cells in G1 in response to
IPTG-induced p16/CDKN2A expression. No significant difference in the
percentage of cells in G1 or S phase of the cell cycle was
observed in AtT20 cells pre- and posttransfection (data not shown). In
parallel with the FACS analysis studies described for clone AtT20/p16+,
the cell populations were also subjected to analysis by Western
blotting for p16 protein and also for the characterization of pRB
phosphorylation status. Figure 4
shows expression of p16 protein in
response to IPTG induction of p16. AtT20 cells failed to express p16
protein; however, in contrast, transfected cells in the presence of 0.2
mM IPTG expressed readily detectable p16 that showed
increased levels of expression in response to higher doses of IPTG (5
mM). Figure 4
also shows for pRB that enforced expression
of p16 is accompanied by a graded shift in the phosphorylation status
of pRB toward its hypophosphorylated form with increasing doses of
IPTG. In parallel with these studies, we also assessed growth by colony
forming efficiency and observed [as shown previously (see Fig. 2
)])
an 85% reduction in colony forming efficiency relative to controls
(data not shown).

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Figure 4. Cell Cycle Arrest Induced by 5 mM IPTG
FACS analysis showing distribution of cells treated without (a) or with
(b) IPTG for 72 h. G1 peak at 64 fluorescence units,
and G2 peak at 128 fluorescence units. In the IPTG-treated
cells approximately 82% of the cells are in G1 as compared
with 60% for the untreated controls (top panel). A
proportion of the cells treated as described in the top
panel were subject to Western blot analysis for human p16
protein and endogenous mouse pRB. For p16 a specific band at 16 kDa
(arrowed) is evident only in cells induced by IPTG. Lane
1, Human pituitary; lane 2, AtT20 before transfection; lanes 35,
clone AtT20/p16 in the absence (3 ) and presence of 0.2 mM
(4 ) and 5 mM (5 ) IPTG (middle panel). For
endogenous pRB a specific band is seen in both induced and uninduced
cells. Relative to the uninduced cells the induced cells show a shift
in the phosphorylation status from the hyperphosphorylated form (ppRb)
toward the active hypophosphorylated species (pRB). Lanes 13 clone
AtT20/p16 in the absence (1 ) and presence of 0.2 mM (2 ) and
5 mM (3 ) IPTG (lower panel). For both p16
and pRb the position of the mol wt markers are shown on the
left of the figures (middle and bottom
panels).
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Reversibility of Enforced G1 Arrest in
AtT20 Cells
To determine whether G1 arrest was reversible, cells
were incubated with IPTG and then allowed to recover as described (see
Materials and Methods). Figure 5
shows that G1 arrest caused
by induction of p16/CDKN2A is reversible. In these stably transfected
cells, the proportion of cells in G1 returns to that of
normal (uninduced) growing cells (
60%) by 72 h following
removal of IPTG. A similar pattern of reversibility in the presence and
absence of 5 mM IPTG was seen over three successive cycles.
This shows that G1 arrest is reversible and that release
from G1 does not alter the cells capacity to undergo
further arrest at G1 after further induction.

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Figure 5. Reversibilty of G1 Arrest in the Cell
Line Stably Transfected with the Inducible p16 Construct (Clone
AtT20/p16+)
The figure shows the percentage of cells in G1 as assessed
by FACS analysis vs. time. Cells were pulsed with IPTG
for 72 h followed by 72 h withdrawal in fresh media and the
cycle was repeated three times. The figure shows that withdrawal of
IPTG results in a decrease in the proportion of AtT20 cells in
G1, whereas rechallenge with IPTG increases the
G1 population. Control cells that are stably transfected
with the inducible p16 construct (clone AtT20/p16+) but were not
treated with IPTG are represented by whereas stably transfected
cells treated with IPTG are represented by . The values represent
the mean and the bars the SD from triplicate
determinations.
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DISCUSSION
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The role of p16/CDKN2A in G1 arrest has been
established in both normal (34) and tumor cell lines (35). A number of
studies have shown that enforced expression of p16/CDKN2A in
transfected cells can inhibit cell proliferation in several tumor types
(24, 36, 37). However, there are instances in which ectopic expression
is without effect. Hengstschlager et al. (38) showed that
growth of pRb+/p16- leukemic cell lines was
not affected by transient overexpression of p16 protein. Transfection
of the originally published p16/CDKN2A sequence has also been shown to
have only a modest and variable effect on cell proliferation in a
number of tumor cell lines (24). In addition, analysis of normal tissue
including colon (21), breast (22), and brain (23) shows that p16/CDKN2A
is not expressed in these tissues, suggesting that alternative
mechanisms are responsible for mediating cell cycle arrest. To
delineate the role of p16/CDKN2A in pituitary tumorigenesis we have
used, as a model system, the mouse corticotroph cell line AtT20 that,
in contrast to normal mouse pituitary, does not express the p16/CDKN2A
gene. Stable p16/CDKN2A transfectants were generated in which
p16/CDKN2A expression was inducible by addition of IPTG. Induction of
p16/CDKN2A gave rise to a profound reduction in colony forming
efficiency and cell proliferation, as assessed by growth curves, that
was accompanied by expression of p16/CDKN2A gene transcript and p16
protein expression as assessed by Western blotting. The mechanism
responsible for the decrease in cellular proliferation was due to a
shift of the cell population into G1 and a progressive
change in the phosphorylation status of pRB toward its
hypophosphorylated (active) form. Thus, these results are consistent
with the in vivo role of p16/CDKN2A at this checkpoint
of the cell cycle. Increasing the dose of inducing agent (IPTG) from
0.2 to 5 mM resulted in a graded increase in p16 protein
levels and concomitant hypophosphorylation of RB proteins. Several
other studies have shown that reexpression of p16/CDKN2A in a variety
of tumor cell lines was sufficient to cause arrest of the cells in
G1 (12, 36, 38, 39, 40, 41, 42). In agreement with these studies,
induction of p16/CDKN2A expression in the AtT20 cell line caused more
than 80% of cells to arrest in G1. Removal of inducing
agent (IPTG) showed that the G1 arrest was reversible over
the three cycles assessed. These results suggest that regulated
(induced) expression in the AtT20 cell line does not lead to
irreversible molecular events that might be lethal or permanent to the
cell population. In this context, in numerous experiments we
found no evidence for apoptosis evident as a sub-G1
population, or by a DNA fragmentation assay (data not shown). In
addition, since cells are released from G1 arrest in the
absence of IPTG, these results suggest that arrest is dependent on
continuous synthesis of p16 protein.
The AtT20 cell line was first described in 1953 as a spontaneously
arising pituitary tumor (43) and has been used by numerous
investigators principally for the study of hormone secretion and
regulation. To our knowledge the accumulated genetic defects
responsible for the initiation of this tumor and generation of its
corresponding cell line are not well characterized. However, in common
with other tumor types, it is unlikely that a single gene defect, in
this case loss of p16/CDKN2A through homozygous deletion, is the only
aberration present. Several key proteins involved in the
G1-S phase transition of the cell cycle, including cyclin
D1 and CDK4, have been characterized, and aberrations in these proteins
in a number of tumor types have been described (44, 45, 46). Enforced
overexpression of CDK4 has been shown to override the ability of
p16/CDKN2A to cause G1 arrest (35). Thus, inappropriate
expression, at this cell cycle checkpoint, of proteins with oncogenic
potential, e.g. cyclins or their kinases, may override
regulators responsible for inhibiting cell cycle progression such as
pRB or p16. In this context, loss of pRB protein (an infrequent event
in human pituitary tumors) cannot be countered by strategies designed
to overexpress p16 (6, 34). Equally, mutations in oncogenes such as
CDK4 or cyclin D1 may render tumors insensitive to p16/CDKN2A-mediated
inhibition (for a detailed discussion see Ref. 42). Despite this lack
of knowledge, with regard to the spectrum of aberrations that impinge
on cell cycle control in AtT20 cells, we show that restoration of
p16/CDKN2A is sufficient to induce cell cycle arrest.
Although we are aware of the dangers of extrapolating from a mouse
model to human pituitary tumors, these findings may have important
implications for novel strategies designed to induce reexpression of
the p16/CDKN2A gene in human pituitary tumors in vivo. Work
by other groups investigating the effects of restoring expression of
tumor suppressor genes such as p53 (47, 48), or RB1 (49, 50), has shown that restoration of these genes is sufficient to inhibit
growth and suppress tumorigenicity, even in cell lines harboring well
characterized multiple genetic aberrations. These studies show that
replacement of a single tumor suppressor gene defect is frequently
sufficient to inhibit tumor growth, and thus restoration of p16/CDKN2A
expression in pituitary tumors may be sufficient to inhibit growth even
against a background of multiple genetic abnormalities.
In several tumor types a direct causal relationship between methylation
of the p16 gene and loss of expression has been demonstrated (21, 25, 26), in which the methylation status of the p16/CDKN2A gene was known.
Since treatment with demethylating agents such as 5-aza-2-deoxycytidine
was sufficient to induce reexpression of p16 in cell lines that had
evidence of p16/CDKN2A methylation, a causal relationship was
established (25, 26, 28). Although for pituitary tumors no such model
exists, it does not immediately preclude studies of primary tumors,
with evidence of methylation within the CpG island of the p16/CDKN2A
gene. However, for several technical reasons these studies are not
possible, in that primary tumors are difficult to propagate in
vitro, demethylating agents are initially cytotoxic, and
reexpression would most likely lead to growth inhibition. Thus, in
these types of studies it would be difficult to assess the mechanism
responsible for reduced growth potential.
In summary we describe the generation of a pituitary tumor cell line in
which p16/CDKN2A gene expression is inducible. Enforced expression in
stable transfectants results in G1 arrest and reduced cell
proliferation. The p16/CDKN2A-mediated G1 arrest did not
lead to a cascade of events that were irreversible and was dependent on
the continued presence of inducing agent. In addition to providing new
insights with regard to the role of p16/CDKN2A in pituitary
tumorigenesis and the functional consequences of restoration of p16
expression, these studies point to novel molecular strategies for
therapeutic intervention, and restoration of growth control in human
forms of this disease.
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MATERIALS AND METHODS
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Cell Culture
The mouse corticotroph tumor cell line AtT20 was obtained from
the European Collection of Cell Cultures Centre for Applied
Microbiology and Research (Porton Down, Salisbury, UK) and cultured in
DMEM supplemented with 15% horse serum, 5% FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate.
Stable Transfection
Cells were seeded at a density of 2 x 105
cells per well and transfected with 15 µl Lipofectin according to the
manufacturers instructions (Life Technologies, Inc.,
Paisley, UK). The AtT20 cell line, which does not express the p16 gene
(see Results), was used as the parent for construction of a
line in which p16 expression is induced by IPTG using the lac-switch
technology as described by the manufacturer (Stratagene,
Cambridge, UK). The amount of lac I expression vector (p3'SS) and the
vector expressing human p16, pOPRSVI.p16 (a kind donation from Dr. A.
Kamb, Myriad Genetics Inc., Salt Lake City, UT), used in each
transfection was 4 µg. Cells were cotransfected for 6 h, after
which culture media were replenished and cells incubated for 48 h
before selection of transformants in 150 µg/ml hygromycin B and 200
µg/ml Geneticin (Life Technologies, Inc.). Ten
transformants were cloned and expression of lac repressor was confirmed
by RT-PCR (see below). The effect of p16/CDKN2A expression on
proliferation of cells was initially determined by in vitro
growth assay (colony forming efficiency). Six clones showed a marked
decrease in proliferation, as assessed by reduction in colony forming
efficiency, in response to p16/CDKN2A induction. Four of the clones
were subject to FACS analysis to determine whether the decrease in
proliferation was due to G1 arrest consistent with a role
for p16/CDKN2A in regulating cell cycle progression (see below).
Colony Forming Efficiency Experiments
The effect of transfected p16 on cell growth was initially
assessed by inhibition of colony forming efficiency, essentially as
described previously (51). Stably transfected cells (5 x
105 per 60-mm dish) were transferred to soft agar medium
consisting of 0.3% noble agar, 30% horse serum, 10% Fetal clone I
(Pierce & Warriner, Chester, UK), 1 mM Na pyruvate, and 2
mM L-glutamine in Hams F-10 media. Cells were
overlaid with cloning medium (30% horse serum, 10% Fetal clone I, 1
mM Na pyruvate, and 2 mM
L-glutamine in Hams F-10 media). Transfected cells in the
absence or presence of IPTG (0.25 mM) were cultured for
72 h, after which microscopic colonies were counted on 60-mm grid
marked petri dishes (Corning Costar Ltd, High Wycombe,
UK). A minimum of 200 colonies were counted in the control samples
(-IPTG) (designated as 100% colony forming efficiency) and compared
with the number of colonies (observed in a grid marked area of equal
size to that of the control) generated from cells treated with varying
doses of IPTG.
Cell Inhibition Assay
The effects of enforced p16 expression on the growth of a stably
transfected clone, designated AtT20/p16+, relative to a clone stably
transfected with a vector lacking the p16 cDNA [AtT20/p16 (-)] was
assessed by growth curve analysis. On the basis of the response seen by
colony forming efficiency (see Fig. 2
) growth curves were
carried out at doses of IPTG shown to have a profound (5
mM) or moderated (0.2 mM) effect or to be
without effect (absence of IPTG) on cell proliferation. Cells were
plated at a density of 1 x 105 per 75-cm2
flask in 10 ml media (see above). Triplicate flasks were used for each
time point. Media were supplemented with IPTG (0.2 and 5
mM) or vehicle alone and replaced at 2-day intervals.
Triplicate individual flasks were sacrificed at each time point (2 day)
and counted, and their viability was determined by trypan blue
exclusion. The experiment was repeated three times.
FACS Analysis
Stably transfected cells at a density of 2 x
106 cells per flask were seeded into tissue culture flasks
and incubated in the absence or presence of 5 mM IPTG in
fresh media for 72 h. After incubation, cells were harvested and
washed with sterile PBS. A proportion of the cells were used for
Western blot analysis (see below) and colony forming efficiency (see
above). The remaining cells were resuspended in 0.2% Triton-X and 50
µM propidium iodide (Sigma Chemical Co., Poole, UK).
Resuspended cells were incubated at room
temperature for 15 min and then stored at 4 C until analysis. FACS
analysis was performed on an EPICS Elite (Coulter Corp., Hialeah FL).
In addition and in parallel with the flow cytometry studies, Western
blot analysis and colony forming efficiency of cells were also
measured. Each experiment was repeated at least three times with
triplicate determinations within each experiment.
Western Blot Analysis
In parallel with the studies described above (FACS analysis),
transfected cells in the absence and presence of IPTG were subject to
Western blot analysis for p16 protein expression and for the
determination of pRB phosphorylation status. Since the p16/CDKN2A
expression vector encoded a human cDNA as a positive control, we
included protein extracted from postmortem derived human pituitaries,
isolated within 12 h of death. Samples were solubilized in 0.5 ml
lysis buffer containing 50 mM Tris-HCL (pH 8), 0.5
mM phenylmethylsulfonylfluoride, 0.02% sodium azide, 0.1%
sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 1.5 µM aprotinin. Samples were
homogenized in a 1-ml glass homogenizer and then centrifuged at
12,000 x g for 10 min at 4 C, and the supernatant was
stored at -70 C until assayed. For p16 expression studies, samples (20
µg) were separated by SDS-PAGE using a 4% stacking and 10%
separating gels in a minigel apparatus along with mol wt markers. After
electrophoresis the gels were rinsed with transfer buffer containing 48
mM Tris base, 39 mM glycine, 0.037% SDS, and
20% methanol. The proteins were subject to electroblotting overnight
on to nitrocellulose paper. After blotting, nonspecific protein binding
was blocked using 5% skimmed milk powder in PBS for 1 h.
Monoclonal antibody directed against human p16/CDKN2A protein (13251A,
PharMingen, San Diego, CA) was diluted 1:200 in PBS and
incubated overnight at 4 C followed by three washes in 0.05% Tween 20
in PBS. The membrane was then probed with sheep antimouse secondary
antibody diluted 1:1000 in PBS. The antibody reaction was revealed by
chemiluminescence detection, according to the manufacturers
recommendations (Pierce & Warriner, Chester, UK). After probing, blots
were stripped and reprobed with antivinculin antibody (Sigma Chemical Co.) to
confirm protein integrity and equal loading. To
investigate the effect of p16 induction on the phosphorylation status
of RB protein, samples (20 µg) were prepared as described above and
electrophoresed for 1 h, 45 min at 150 V, using a 38% gradient
Tris-Acetate precast polyacrylamide gel, (Novex, San
Diego, CA). Samples were electrotransferred from the gel to a
nitrocellulose membrane for 1 h, 15 min, at 30 V. After blotting,
nonspecific protein binding was blocked using 1% BSA in PBS for 1
h. Monoclonal antibody directed against both the hypo- and
hyperphosphorylated forms of the RB protein (G3245
PharMingen) was diluted 1:200 in PBS and incubated with
the membrane at 4 C overnight followed by three washes in 0.05% Tween
20 in PBS. RB protein was visualized by chemiluminescence, as described
above.
Reversibility Studies
To determine whether the effect of p16/CDKN2A induction was
reversible, cells were seeded as above and incubated in the absence or
presence of 5 mM IPTG for 3 days, after which cells were
harvested and an aliquot taken for FACS analysis. The remaining cells
were then washed three times in sterile PBS and cultured for an
additional 3 days in the absence of IPTG, after which cells were again
harvested and a sample analyzed by flow cytometry. This cycle was
repeated three times to assess whether cells could recover from the
enforced G1 arrest caused by induction of p16/CDKN2A
expression.
Reverse Transcription (RT)
Total RNA (5 µg) was isolated from 3 x 106
cells lysed in guanidinium isothiocyanate as previously described (52).
cDNA synthesis was achieved using a commercially available kit,
essentially as described by the manufacturer (Life Technologies, Inc.).
PCR Amplification of p16/CDKN2A and RB1
Expression of the p16/CDKN2A transcript in the AtT20 cell line
was determined by PCR amplification as follows. cDNA was amplified with
primers specific for the mouse p16/CDKN2A gene (sense
5'-GCTGCAGACAGACTGGCCA-3'; antisense 5'-GTCCTCGCAGTTCGAATCTG-3'; PCR
amplicon 189 bp, annealing temperature, 53 C). Human p16/CDKN2A (sense,
5'-ATGGAGCCTTCGGCTGACT-3', antisense, 5'-GGCGCAGTTGGGCTCC-3', PCR
amplicon 190bp, annealing temperature, 55 C). Human p16/CDKN2A specific
primers were used to confirm expression of human p16/CDKN2A in the
mouse cell line after generation of stable transfectants. The
housekeeping gene Porphobilinogen Deaminase (PBGD) was coamplified in a
multiplex PCR reaction, with mouse-specific primers (sense
5'-CGTCGGCTTCTGCAGACACC-3', antisense 5'-GGCTCTTACGGGTGCCCA-3' PCR
amplicon 150 bp, annealing temperature, 55 C). Confirmation of
RB1 transcript expression in the AtT20 cell line was
assessed using mouse (sense 5'-GAGCTTGGCTAACTTGGG-3', antisense,
5'-GCATTATCAACCTTGGTACT-3', PCR amplicon 220 bp, annealing temperature,
56 C). All oligonucleotides were designed to amplify specific sequences
based on Genome Data Base information. PCR reactions were carried out
in 25 µl volumes with 1.5 mM MgCl2, 200
µM each of dATP, dGTP, dTTP, and dCTP, 2 pmol of each
primer template DNA, and 1 U Taq DNA polymerase.
Amplification was facilitated by the addition of 3.6% formamide to the
reaction buffer. PCR products were resolved on 8% polyacrylamide gels,
fixed in 10% methanol-0.5% acetic acid for 6 min, and then incubated
in 0.1% aqueous silver nitrate for 15 min. After two brief washes in
distilled water, PCR products were visualised by development in 1.5%
sodium hydroxide-0.1% formaldehyde.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. A. Kamb for the pOPRSVIp16 vector. In
addition we wish to thank Dr. Paul Hoban for his critical reading of
the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. W. E. Farrell, Centre for Cell and Molecular Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke-on-Trent, United Kingdom ST4 7QB.
This work was supported in part by the Association for International
Cancer Research (AICR) and West Midlands Regional Health
Authority.
Received for publication April 1, 1999.
Revision received August 3, 1999.
Accepted for publication August 4, 1999.
 |
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