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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (80–82%). 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1aGo). Fig 1aGo 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. 1bGo). 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.

 
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. 1cGo). 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. 2Go. Varying the dose of IPTG from 0.2–5 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 0.2–5 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.

 
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 3Go shows that for the clone AtT20/p16+ growth in the presence of 5 mM IPTG (Fig. 3aGo) 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. 3bGo) showed an intermediate reduction in cell proliferation, consistent with the partial decrease in growth evident in the colony forming efficiency studies (Fig. 2Go). Figure 3cGo 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. 3cGo).



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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. 1Go and 2Go). 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.

 
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 4Go 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 1Go 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 4Go 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 4Go 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. 2Go)]) 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 3–5, 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 1–3 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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Table 1. Proportion of AtT20 Stable Clones in G1 in the Presence or Absence of IPTG

 
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 5Go 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 {blacksquare}. The values represent the mean and the bars the SD from triplicate determinations.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 (0.2–5 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. 2Go) 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 3–8% 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 (G3–245 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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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