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Journal of Virology, September 2008, p. 8383-8391, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00348-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Changes in p19^Arf Localization Accompany Apoptotic Crisis during Pre-B-Cell Transformation by Abelson Murine Leukemia Virus

Rebekah Stackpole Zimmerman^1,2, and Naomi Rosenberg^1,2,3*

Genetics Graduate Program, Sackler School of Graduate Biomedical Sciences,¹ Departments of Pathology,² Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111³

Received 18 February 2008/ Accepted 13 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Transformation by Abelson murine leukemia virus (Ab-MLV) is a multistep process in which growth-stimulatory signals from the v-Abl oncoprotein and growth-suppressive signals from the p19^Arf-p53 tumor suppressor pathway oppose each other and influence the outcome of infection. The process involves a proliferative phase during which highly viable primary transformants expand, followed by a period of marked apoptosis (called "crisis") that is dependent on the presence of p19^Arf and p53; rare cells that survive this phase emerge as fully transformed and malignant. To understand the way in which v-Abl expression affects p19^Arf expression, we examined changes in expression of Arf during all stages of Ab-MLV transformation process. As is consistent with the ability of v-Abl to stimulate Myc, a transcription factor known to induce p19^Arf, Myc and Arf are induced soon after infection and p19^Arf is expressed. At these early time points, the infected cells remain highly viable. The onset of crisis is marked by an increase in p19^Arf expression and a change in localization of the protein from the nucleoplasm to the nucleolus. These data together suggest that the localization and expression levels of p19^Arf modulate the effects of the protein during oncogenesis and reveal that the p19^Arf-mediated response is subject to multiple layers of regulation that influence its function during Ab-MLV-mediated transformation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Abelson murine leukemia virus (Ab-MLV) expresses a potent oncogene that encodes the v-Abl nonreceptor protein tyrosine kinase (reviewed in reference 31). Infection of bone marrow cells in vitro induces transformation of cells resembling early pre-B-cell lymphocytes, and mice infected with the virus develop a rapid lymphoma made up of similar types of cells. Despite the strong oncogenic signals provided by v-Abl, transformation is a multistep process. Oncogenesis begins with an early polyclonal proliferative phase both in vivo and in vitro but culminates with the emergence of clonal, malignant cells (7, 38, 45, 46). Dissecting the process in vitro has revealed that the proliferative phase is followed by an apoptotic crisis, during which cells that have acquired changes affecting the p53 pathway are selected (26, 38, 39). Fully transformed cells typically either acquire p53 mutations or downregulate expression of the p19^Arf tumor suppressor, a protein that regulates p53 through effects on Mdm2 and other molecules (reviewed in reference 6). As is consistent with these data, cells from mice lacking either p53 or Arf bypass the crisis phase of transformation (26, 39).

The way in which p19^Arf expression influences Ab-MLV transformation is not completely understood. The protein interacts with Mdm2, a ubiquitin ligase that targets p53 for proteosome-mediated degradation (17, 19). The ability of p19^Arf to inhibit Mdm2 leads to stabilization of p53, which triggers downstream events such as apoptosis and cell cycle arrest. Although p19^Arf is localized to the nucleolus in many cell types and in some circumstances appears to interact with Mdm2 within this structure (43, 44), under conditions of stress and DNA damage these interactions do not require nucleolar localization (14, 16). Indeed, other data indicate that nucleolar p19^Arf is bound by nucleophosmin (NPM), an interaction that stabilizes p19^Arf and inhibits its ability to activate p53 through effects on Mdm2 (4, 13). Thus, some experimenters argue that nucleolar p19^Arf plays a role in p53 activation, while others propose that p19^Arf can be sequestered in the nucleolus, through interactions with NPM, to protect the cells from the proapoptotic effects of the molecule. In addition, p53-independent functions of p19^Arf are well documented (20, 37, 42) and basal levels of p19^Arf present in independence of oncogenic or stress signals appear to influence ribosome biogenesis and growth (1, 36) in at least some cell types.

Despite strong evidence that p19^Arf expression is important during Ab-MLV transformation, the mechanisms regulating the protein during the transformation process are not well understood. For example, increased expression of c-Myc, known to induce upregulation of Arf expression (48), has been reported to occur rapidly in response to v-Abl expression (22, 47, 49). However, even though v-Abl expression occurs very soon after virus integration, previous studies have first detected p19^Arf expression about 2 weeks postinfection, coincident with the onset of the crisis phase of transformation (26). Thus, despite the documented ability of v-Abl to upregulate c-Myc expression (47), the delay in crisis, an event known to require p19^Arf expression (26), indicates that Arf expression and function are regulated by additional mechanisms early in the infection and transformation process. Given the ability of a range of oncogenic signals to stimulate Arf expression, these regulatory circuits may play a role in the pathogenesis of other malignant diseases. To understand these mechanisms, we examined p19^Arf expression throughout transformation. These studies revealed that p19^Arf is expressed prior to crisis induction but that changes in p19^Arf levels, along with a shift in localization from the nucleoplasm to the nucleolus, occur coincidently with apoptotic crisis. These changes occur in a setting where NPM expression is largely cytoplasmic, indicating that p19^Arf function is modulated by several mechanisms that affect its function during Ab-MLV-induced transformation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells, viruses, and mice. Ab-MLV-transformed pre-B cells were grown in RPMI 1640 medium as described previously (26). To recover uninfected pre-B-cell lymphocytes, whole bone marrow cells were stained with antibodies directed against B220 and CD43 (BD Pharmingen) and sorted using a MoFlo instrument. Ab-MLV stocks were prepared using either the pMIG vector (40, 41), pMIA, a modified version of pMIG in which v-Abl sequences were inserted downstream of the internal ribosome entry site (2), or P120-Arf, a pMIA vector in which the HA-Arf fragment from pCI-HA-Arf (25) was inserted into pMIA at the EcoRI site upstream of the internal ribosome entry site (Fig. 1A). To produce virus, 293T cells were transfected with plasmids containing viral sequences as previously described (18). In some experiments, virus was prepared using ANN-1/Cl2, an Ab-MLV-P120-transformed nonproducer cell line rescued with Moloney murine leukemia virus (29). pMIG viruses were subjected to titration by infecting NIH 3T3 cells with serial dilutions of virus and analyzing the frequency of green fluorescent protein (GFP)-positive cells by flow cytometry 24 h later. pMIA viruses were subjected to titration using real-time PCR as previously described (2). For transformation assays, bone marrow cells were harvested and infected using the standard liquid transformation assay (18, 41); cultures were scored as transformed when the density of the rapidly dividing lymphoid cells reached 2 x 10⁶ cells per ml. The Arf–/– mice were maintained by mating heterozygous animals originally obtained from a single breeding pair of Arf+/– animals on a mixed C57BL/6-129SvJ background (12). The mice were backcrossed to C57BL/6 mice for seven generations prior to use in experiments. BALB/cJ mice were originally obtained from The Jackson Laboratory and maintained in our breeding colony.

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FIG. 1. Expression of p19Arf can inhibit transformation in pre-B cells. (A) A schematic diagram of the P120-Arf and P120 viruses. IRES, internal ribosome entry site; LTR, long terminal repeat. (B) Lanes 1 to 3, 293T cells were transfected with 15 µg of viral DNA encoding either P120-Arf or P120 or were subjected to mock transfection; lysates were prepared 48 h posttransfection. Lanes 5 to 12, lysates were prepared from either control cell lines (p53 mutant and Ink4a/Arf null) or cell lines transformed with the Ab-MLV P120 strain, the Ab-MLV P160 strain (lane 6), or the P120-Arf virus and lysates were analyzed using Western blotting with antibodies against Gag/v-Abl (3) and p19Arf.

RNA isolation and PCR. RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer's instructions; 1 µg was reverse transcribed using Superscript II reverse transcriptase (Invitrogen), 1 µM random hexamer primers (Invitrogen), 40 U of RNasin (Promega), 4 mM dithiothreitol (Invitrogen), and 2.5 mM deoxynucleoside triphosphates; and the cDNAs were amplified at 25°C for 10 min, 42°C for 50 min, and then 70°C for 15 min. cDNA was detected using quantitative real-time PCR and an Opticon thermocycler (MJ Research), and the data were analyzed using Opticon Monitor v3.1 software. All reactions were performed in triplicate with reaction mixtures containing 1 ng cDNA, 1x Sybr green PCR Master Mix (Applied Biosystems), and 0.8 µM primer (each). All reaction mixtures were incubated at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and either 59°C for 1 min and 81°C for 1 s (v-abl, Myc, Dmp1, and Bmi-1 amplification) or 58°C for 1 min and 81°C for 1 s (amplification of all other genes). Primer sequences are available upon request.

Immunofluorescence staining. Cells were centrifuged onto microscope slides by use of a Cytospin instrument (Shandon) and fixed in 3% formaldehyde for 10 min. The slides were treated with 0.1% Triton X-100 for 10 min and then with staining buffer (phosphate-buffered saline supplemented to contain 10% fetal calf serum). p19^Arf (Abcam [catalog no. ab80] or Santa Cruz [5-C3-1]), fibrillarin (Abcam [ab5821]), and NPM (Abcam [ab15440]) antibodies were diluted in 0.5% fetal calf serum and incubated on the slides for 1 h in a humidified chamber. The slides were washed in staining buffer and reacted with Alexa 594-conjugated antibodies (Molecular Probes) for 30 min. After washings in staining buffer, the slides were incubated with 300 nM 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 5 min, washed, and mounted. The slides were evaluated using an Eclipse 6400 microscope (Nikon) equipped with Spot Advanced software (Diagnostic Instruments). At least 100 cells were examined in each preparation. ImageJ software (http://rsb.info.nih.gov/ij/) was used to quantitate the fluorescence intensity of each cell.

Protein analysis. Cell lysates were prepared as described previously (2), and 30 µg of protein was fractionated through a sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (U.S. Biochemicals). The membrane was treated with primary and secondary antibodies as previously described (2). Primary antibodies used included anti-p19^Arf (catalog no. ab80; Abcam), anti-Gag/v-Abl (H548) (3), and anti-NPM (ab15440; Abcam).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of p19^Arf can inhibit transformation in pre-B cells. Previous work has shown that p19^Arf induces apoptosis in fully transformed pre-B cells that retain wild-type p53 and that primary transformants derived from Ink4a/Arf or Arf null mice bypass the crisis phase of the transformation process (26, 30). To determine whether expression of p19^Arf during primary transformation affects this stage of the process, bone marrow cells were infected with virus expressing v-Abl or virus expressing both p19^Arf and v-Abl (Fig. 1A). This virus expressed p19^Arf at levels two- to threefold higher than those recovered from fully transformed cells that expressed mutant p53 (Fig. 1B), the type of Ab-MLV transformant that expresses the highest levels of p19^Arf (26). When the cells were plated in liquid medium and monitored for transformation, all cultures infected with virus expressing v-Abl became transformed within the standard 10-day period (Table 1). In contrast, only 4 of 16 cultures infected with P120-Arf gave rise to transformed cells. These transformants emerged after a slightly longer 11- to 14-day incubation period postinfection. Analyses of all four of these transformants by use of Western blotting (Fig. 1B) revealed that none of the cells in these cultures continued to express p19^Arf, suggesting that the cells that gave rise to these transformants had lost the ability to express this protein. These data indicate that expression of p19^Arf can inhibit transformation at the primary transformation phase.

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TABLE 1. Expression of p19Arf can inhibit transformation by Ab-MLVa

The Arf gene is expressed soon after v-Abl expression. Expression of p19^Arf inhibits primary transformation, and yet pre-B cells from wild-type mice and those from Arf null mice are equally susceptible to primary transformation. As is consistent with this finding, earlier work indicated that p19^Arf expression coincides with the onset of the crisis phase of transformation (26). These data could indicate that expression of endogenous p19^Arf is repressed during the early phases of transformation, suggesting a previously unrecognized mode of Arf regulation. Such an idea is countered by literature indicating that Myc, a gene known to be upregulated in expression by v-Abl in at least some cell types (22, 47, 49), can induce Arf expression (48). To determine how expression of these genes is affected by v-Abl expression in pre-B cells, bone marrow cells were infected with a virus that expresses v-Abl and GFP and the infected cells were recovered by flow cytometry 6, 8, and 10 days postinfection. The day 6 time point was the earliest time postinfection that sufficient numbers of infected cells could be recovered for analysis and represents a time when infected cells are healthy and expanding rapidly. cDNAs were prepared from these cells and from uninfected B220⁺ CD43⁺ bone marrow cells, the B-lineage population that contains cells susceptible to v-Abl-mediated transformation (M. Gunthart and N. Rosenberg, unpublished data). Real-time PCR assays were used to monitor expression of v-abl, Myc, and Arf; copies of 18S RNA were used to standardize each of the samples (Fig. 2). As expected, the infected cells expressed high levels of v-abl RNA, and, consistent with the ability of v-Abl to stimulate Myc (47), levels of Myc RNA were increased more than 10-fold compared to those observed with the normal, uninfected target cell population. In addition, compared to the control sample results, 100-fold-higher levels of Arf RNA were recovered from the infected samples. These data indicate that Arf expression occurs as primary transformants are expanding during the initial phases of transformation.

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FIG. 2. v-abl, Myc, and Arf are induced early in transformation. RNA was harvested from uninfected B220+ CD43+ cells (uninf.) and infected cells 6, 8, and 10 days after infection with Ab-MLV and reverse transcribed with random hexamer primers. Real-time PCR using primers specific for v-abl (A), Myc (B), and Arf (C) was performed, and results were normalized to 18S RNA results. Error bars represent the standard deviations of the means of the results of three independent PCRs from one reverse transcription. The data are representative of values obtained for at least two independent infections in which cDNAs from two independent reverse transcriptions were analyzed in triplicate.

Expression of Arf increases about 10-fold during crisis. Although Arf is expressed early in transformation, these levels of expression may not be sufficient for a biological response. To examine this possibility, cDNA was prepared from primary transformants as they entered crisis at 17 days postinfection and during the crisis phase at 21 days postinfection. Control samples included the normal cells used as described above and cDNAs from a panel of established, fully transformed cells. The latter group included transformants prepared from Ink4a/Arf or p53 null mice, transformants that retained wild-type p53 and did not express p19^Arf protein, and transformants that expressed mutant p53 and readily detectable p19^Arf.

PCR analyses revealed that levels of v-abl and Myc RNA were generally within the same range as those found in fully transformed cells and similar to those recovered during the early phases of primary transformation (Fig. 3). In contrast, levels of Arf expression increased about 10-fold in crisis cells. These levels were comparable to those recovered from fully transformed cells that express mutant p53 and abundant p19^Arf but were about 100-fold higher than those recovered from fully transformed cells that retained wild-type p53 and expressed very little p19^Arf protein. These data indicate that steady-state levels of Arf RNA are modulated throughout the transformation process. The largest increase occurred soon after infection, with additional increases as the transformation process proceeded. Cells that retained wild-type p53 and were fully transformed exhibited downregulated expression.

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FIG. 3. Arf mRNA expression increases as cells enter crisis. RNA was harvested from five different infected cultures 17 and 21 days after infection with Ab-MLV (gray bars) as well as from cultures of six different established cell lines of the indicated genotypes (black bars). The RNA was reverse transcribed with random hexamer primers, followed by real-time PCR using primers specific for v-abl (A), Myc (B), and Arf (C). Results were normalized to 18S RNA results. Error bars represent the standard deviations of the means of the results of three PCRs from one reverse transcription. The data are representative of values obtained for at least two independent infections in which cDNAs from two independent reverse transcriptions were analyzed in triplicate.

Several genes regulating the Arf and p53 pathways display small changes in expression during transformation. Analyses of Arf RNA levels revealed changes throughout transformation. To determine whether changes in expression of genes involved in the p19^Arf or p53 pathways are altered during transformation, real-time PCR was used to examine expression of Dmp1, Bmi-1, p53, and Bax (Fig. 4). Dmp1 can activate Arf in a Myc-independent fashion (33), and Bmi-1 (10) encodes a protein that can repress expression from the Ink4a/Arf locus (32). Bax is one of several proapoptotic proteins that are stimulated by p53 (21). Analyses of expression of Dmp1 revealed that infected cells expressed about six- to sevenfold more Dmp1 than cells in the uninfected target cell population, an amount that increased severalfold for fully transformed cells independently of p19^Arf status. Changes in levels of Bmi-1 RNA were more subtle, but somewhat higher levels of Bmi-1 RNA were detected in fully transformed cells. p53 expression was elevated in newly infected cells compared to the results seen with normal cells but was only severalfold higher in samples tested during crisis. Bax RNA was induced about 100- to 1,000-fold throughout the transformation process, an increase that correlated with infection but not with crisis induction. These data suggest that infection alters the expression of other genes involved in the p53 and p19^Arf pathways but do not reveal a pattern that correlates with crisis.

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FIG. 4. Genes involved in the Arf-p53 pathway show subtle changes in expression during transformation. Real-time PCR was performed using the same cDNAs described for Fig. 2 and 3 with primers specific for Dmp1 (A), Bmi-1 (B), p53 (C), and Bax (D), and the results were normalized to 18S RNA results. Error bars represent the standard deviations of the means of the results of three PCRs from one reverse transcription. The data are representative of values obtained for at least two independent infections in which cDNAs from two independent reverse transcriptions were analyzed in triplicate. uninf., uninfected.

p19^Arf protein is expressed in primary transformants but is not always found in the nucleolus. RNA analyses demonstrate that Arf is expressed soon after infection, prior to the time points where the protein was detected in previous studies (26). Because those analyses were conducted using early-generation antibodies and Western blotting, p19^Arf expression was revisited using more-sensitive reagents and immunofluorescence staining. Individual clonal primary transformants were compared at 10 days postinfection to transformants that expressed a mutant form of p53 and therefore produced high levels of p19^Arf and to transformants derived from Ink4a/Arf null mice. As expected, no cells in the Ink4a/Arf null transformant were stained, whereas all cells in the p53 mutant cell line reacted with the antibody and expressed readily detectable p19^Arf that colocalized with fibrillarin, a protein that localizes to the nucleolus (27) (Fig. 5A). Analyses of the primary transformants revealed p19^Arf-positive cells in all 26 isolates, with the frequency of positive cells ranging from about 30% to more than 95% (Fig. 5B and C). However, in contrast to the results seen with transformants expressing mutant p53, p19^Arf staining was diffuse throughout the nucleoplasm in many primary transformants (Fig. 5B). In some populations, less than 25% of the cells expressed nucleolar p19^Arf, whereas in others, over 75% of the cells expressed p19^Arf in the nucleolus (Fig. 5C). No differences in cellularity or viability could be correlated to these differences, a finding consistent with the variable kinetics that characterizes the onset and resolution of crisis (26, 39). Thus, p19^Arf expression per se is not sufficient to induce apoptosis in early transformation, and localization of p19^Arf may affect the ability of cells to tolerate expression of the protein.

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FIG. 5. Some primary transformants do not express nucleolar p19Arf. (A) Control cell lines expressing mutant p53 or derived from Ink4a/Arf null mice were costained with antibodies against p19Arf and fibrillarin, a nucleolar marker (27). (B) Primary transformants and control cell lines were stained with antibodies directed against p19Arf and with DAPI and examined using a fluorescence microscope. The images shown are representative of analysis of at least 100 cells in each preparation. Primary transformants were examined 10 days postinfection. (C) The frequency of cells expressing p19Arf in the nucleolus or in the nucleoplasm was determined by examining the staining pattern in control cells (left panel) and in primary transformants (right panel). A minimum of 100 cells were scored for each sample. Examples of results obtained with primary transformants examined 10 days postinfection and derived from four independent experiments are shown. Black bars indicate nucleolar localization; white bars indicate localization in the nucleoplasmic region.

Localization of NPM is altered in cells infected with Ab-MLV. NPM, a protein found in the nucleolus in most cells (reviewed in reference 8), influences the localization and function of p19^Arf (4, 13). To explore the possibility that NPM might influence p19^Arf localization during Ab-MLV transformation, Western blotting was used to monitor NPM protein expression at multiple time points during transformation. Both primary transformants and established transformants expressed readily detectable NPM (Fig. 6A). However, NPM expression was not detected in uninfected B220⁺ CD43⁺ cells, indicating that v-Abl expression upregulates expression of NPM. When localization of NPM was examined using immunofluorescence staining, intense nucleolar staining was detected in unfractionated bone marrow cells but, consistent with the Western analysis results, uninfected B220⁺ CD43⁺ cells were not stained with anti-NPM antibodies (Fig. 6B). Primary transformants were uniformly positive for NPM, but the molecule was expressed almost exclusively in the cytoplasm. Samples from cells harvested 17 days after infection, during the crisis phase, continued to express cytoplasmic NPM. At that time point, some NPM was detected in the nucleus, but it was not localized to distinct structures that resembled the nucleolar region stained in p53 mutant cells expressing p19^Arf. Although these data suggest that v-Abl alters the expression and localization of NPM, they also indicate that NPM localization may not directly influence p19^Arf function during transformation.

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FIG. 6. Ab-MLV transformants express NPM in the cytoplasm. (A) NPM expression was examined by Western blotting using uninfected B220+ CD43+ pre-B cells (Uninfected), primary transformants examined 10 days postinfection, and a panel of fully transformed cell lines of the indicated genotypes. The blot was stripped and reprobed with antibodies against β-actin to control for loading. (B) Whole bone marrow, uninfected B220+ CD43+ cells, primary transformants, and cells in the crisis phase of transformation were stained with antibodies against NPM and with DAPI and examined by immunofluorescence microscopy. A minimum of 100 cells were examined for each sample.

Nucleolar p19^Arf localization increases during crisis and correlates with viability. All of the cells in the primary transformants examined as described for Fig. 5 were used for the staining experiment. Thus, although our earlier work revealed that only a fraction of primary transformants go on to become fully transformed (26, 38, 39), we could not determine whether the primary transformants with the highest frequency of nucleolar p19^Arf-positive cells were those that would succumb during crisis. To examine this possibility, primary transformants were expanded such that sufficient numbers of cells were available for monitoring changes in p19^Arf localization and viability during the crisis period (Fig. 7). Analyses of eight individual primary transformants for p19^Arf expression and localization and for viability during crisis revealed that the frequency of cells expressing nucleolar p19^Arf increased during this period and that the increase coincided with a decrease in viability. At day 14 postinfection, a time when more than 50% of the cells were viable in most of the cultures, p19^Arf was found in the nucleolus in fewer than 50% of the cells in most of the populations. By 22 days postinfection, a time at which viability had decreased in all cultures and two of the cultures contained too few cells to analyze, the majority of cells in the population expressed p19^Arf in the nucleolus.

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FIG. 7. The percent of cells with nucleolar p19Arf increases as cells enter crisis. Primary transformants were plated in liquid medium and analyzed during the outgrowth and crisis period. (A) Cells were stained with antibodies directed against p19Arf 14, 17, 21, and 22 days after infection and examined by immunofluorescence microscopy for the frequency of cells expressing p19Arf in the nucleolus and in the nucleoplasm. At least 100 cells were examined for each sample. The black bars indicate nucleolar localization; the white bars indicate nucleoplasmic localization; an asterisk indicates that too few cells were available for analysis. (B) The viability of cells in the cultures examined as described for panel A was determined by counting trypan blue-stained cells with a hemocytometer.

Levels of nucleolar p19^Arf increase during crisis. Analyses of Arf RNA levels revealed that increased expression was also a feature of cells in crisis, raising the possibility that protein levels also increase during this period. In addition, nucleolar localization stabilizes p19^Arf (15, 28). To examine levels of nucleolar p19^Arf in individual cells, immunofluorescence images were analyzed using ImageJ, a software package that calculates the intensity of fluorescence in individual cells. The range of values obtained for individual cells was used to calculate a percentile rank for each sample, thereby allowing comparison of values across experiments. The percentile rank data were then used to determine the mean percentile rank for samples analyzed during the crisis period (Table 2). All eight of the samples showed a 1.4- to 2-fold increase in the intensity of nucleolar p19^Arf from days 21 to 22. All of the transformants succumbed to crisis except for RS3-35, which was able to establish by day 35 and highly expressed p19^Arf in the nucleolus because of a mutation in p53. These results suggest that the localization and expression levels of p19^Arf might be important for the induction of crisis.

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TABLE 2. Nucleolar p19Arf expression levels increase as cells enter crisis

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have taken advantage of the multistep process of Ab-MLV transformation to demonstrate that p19^Arf is subject to multiple layers of regulation during oncogenesis. The sequence of events we have documented reveals that the p19^Arf response to a single oncogenic signal from the v-Abl protein is modulated in a complex fashion at multiple levels throughout the transformation process. Regulatory mechanisms that influence RNA expression levels and those that affect subcellular localization modulate p19^Arf function in response to oncogenic signals from v-Abl and affect the outcome of Ab-MLV infection. As is consistent with the ability of v-Abl to activate Myc expression (47, 49) and the ability of Myc signaling to stimulate Arf expression (48), Arf RNAs appear soon after infection, probably coincidently with accumulation of the v-Abl protein. However, this expression is not sufficient to trigger the p53 response required for apoptosis and crisis induction, events that require p19^Arf and occur at a later stage in transformation (26, 38, 39). A shift in p19^Arf subcellular localization from the nucleoplasm to the nucleolus, a change accompanied by increased levels of p19^Arf, provides a second critical level of regulation. This change correlates strongly with the onset and severity of the p53-dependent apoptotic crisis stage of transformation (39), suggesting that nucleolar localization is required for this phase of the process.

The way nucleolar localization affects p19^Arf function is not fully understood. Increased protein stability is one feature of nucleolar p19^Arf (15, 28), and the higher levels of the molecule that are detected during crisis may in part reflect increased nucleolar localization. However, the results obtained with some models indicate that p19^Arf is not able to activate p53 when localized to the nucleolus (13) and that relocalization of the Arf-Mdm2 complex to the nucleolus is not required to induce p53-mediated growth arrest in cells (14, 16). Nonetheless, during Ab-MLV transformation, localization of p19^Arf to the nucleolus correlates well with the decline in the viability of the cells, suggesting that the protein can trigger p53-dependent apoptosis from this location within the cell. This interpretation is consistent with work showing that increased expression of Arf leads to the nucleolar colocalization of Arf and Mdm2 and activation of p53 (44). In addition, a mutant form of Arf that retains the ability to bind Mdm2 but cannot move into the nucleolus failed to activate p53 in these studies (44).

Modulation of Arf RNA levels is one way in which p19^Arf levels are regulated during transformation. Levels of Arf RNA are 100-fold higher during primary transformation than in uninfected pre-B cells and decrease dramatically in fully transformed cells that retain wild-type p53. These changes do not correlate with changes in steady-state levels of Myc or v-abl RNAs or with changes in steady-state levels of Bmi-1 and Dmp1 RNAs. The proteins encoded by those latter two genes can be regulated at the posttranscriptional level (reviewed in references 9 and 24), a possibility that was not addressed in our studies. Alternatively, other factors may regulate expression in pre-B cells undergoing Ab-MLV transformation. Indeed, even regulation by Myc, a well-documented Arf regulator, may not be the only or even the dominant way in which v-Abl signals to Arf. Wild-type p53 can suppress Arf expression (34). As is consistent with this finding, established cell lines that retain wild-type p53 express very low levels of p19^Arf and those that express mutant p53 express the highest levels of p53 detected in Ab-MLV transformants.

Changes in NPM expression have not been linked to abl oncogene expression, and the mechanisms by which Ab-MLV infection influences NPM expression and localization during transformation require additional study. NPM expression patterns in B-lymphocyte populations have not been reported. However, many normal cells express low to undetectable levels of NPM, and increased levels can be detected in malignant cells. For example, normal prostate tissue does not express NPM but expression is readily detectable in malignant prostate cancer cell lines (35) and expression of NPM in colorectal tumors is much higher than that observed in the nonneoplastic mucosa (23). Changes in NPM localization of malignant cells have also been reported. One study of cells from patients with acute myelogenous leukemia identified a subset of cells that expressed cytoplasmic NPM as a consequence of mutation affecting the sequences encoding the carboxyl terminus of the protein (5). However, in the transformants studied here, NPM is present in cytoplasm very early in transformation at a time when cells have undergone only 15 to 20 cell divisions, making mutation an unlikely mechanism. Indeed, even in cases in which the selection of cells with a p53 mutation is accelerated by the absence of a functional mismatch repair pathway, a longer period of time is required for emergence of cells with the mutation (11).

Our ability to examine expression of endogenous p19^Arf in cells expressing an oncogenic virus that has undergone biological selection as a transforming agent likely played a key role in uncovering the various regulatory steps that modulate p19^Arf. This approach contrasts with models in which transfection was used to overexpress p19^Arf or other molecules involved in its regulation. Even in the Ab-MLV model, overexpression of p19^Arf blocks transformation. However, cells from Ink4a/Arf or Arf null mice transform at the same frequency as cells from wild-type mice (30), indicating that endogenous levels of p19^Arf do not affect this stage of transformation. The results of our studies thus demonstrate that critical regulatory circuits influencing the outcome of infection with oncogenic viruses and presumably affecting other oncogenic insults can be revealed when studied in the context of normal expression levels. In this regard, the Ab-MLV transformation system provides a rich model for use in uncovering the mechanisms that control the way p19^Arf and p53 influence oncogenesis.

 
We are grateful to John Coffin for assistance with calculations related to fluorescence intensity and to Volkan Gunduz for assistance in constructing some of the vectors used in these experiments.

R.S.Z. was supported in part by grant T32 CA065441; support by grant CA33771 from the National Cancer Institute is also acknowledged.

 
* Corresponding author. Mailing address: Sackler 814, Tufts Medical School, 136 Harrison Avenue, Boston, MA 02111. Phone: (617) 636-2143. Fax: (617) 636-0337. E-mail: naomi.rosenberg{at}tufts.edu

Published ahead of print on 25 June 2008.

Present address: Harvard-Partners Center for Genetics and Genomics, Cambridge, MA 02139.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, September 2008, p. 8383-8391, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00348-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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