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Journal of Virology, June 2003, p. 6208-6215, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6208-6215.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Absence of p53 Complements Defects in Abelson Murine Leukemia Virus Signaling

Indira Unnikrishnan^1, and Naomi Rosenberg^1,2*

Department of Pathology,¹ Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111²

Received 30 December 2002/ Accepted 11 March 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The v-Abl protein encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells via a two-stage process. An initial proliferative phase during which cells with limited tumorigenic potential expand is followed by a crisis period marked by high levels of apoptosis and erratic growth. Transformants that survive this phase emerge as fully malignant cells and usually contain mutations that disable the p53 tumor suppressor pathway. Consistent with the importance of p53 in this process, pre-B cells from p53 null animals bypass crisis. Thus, the transformation process reflects a balance between signals from the v-Abl protein that drive transformation and those coming from the cellular response to inappropriate growth. One prediction of this hypothesis is that Ab-MLV mutants that are compromised in their ability to transform cells may be less equipped to overcome the effects of p53. To test this idea, we examined the ability of the P120/R273K mutant to transform pre-B cells from wild-type, p53 null, and Ink4a/Arf null mice. The SH2 domain of the v-Abl protein encoded by this mutant contains a substitution that affects the phosphotyrosine-binding pocket, and this mutant is compromised in its ability to transform NIH 3T3 and pre-B cells, especially at 39.5°C. Our data reveal that loss of p53 or Ink4a/Arf locus products complements the transforming defect of the P120/R273K mutant, but it does not completely restore wild-type function. These results indicate that one important transforming function of v-Abl proteins is overcoming the effects of a functional p53 pathway.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abelson murine leukemia virus (Ab-MLV) transforms cells by expressing the v-Abl oncoprotein (21). Despite the presence of a strong viral oncogene, pre-B-cell transformation in vivo and in vitro is a multistep process (10, 18, 32, 33). When bone marrow populations are infected, pre-B cells undergo an initial proliferative phase called primary transformation, which is followed by a period of apoptosis and erratic growth called crisis. Some clones of primary transformants succumb to crisis, and others emerge as fully malignant cell lines (18, 26, 27). About half of the cell lines that survive acquire a mutation that renders the p53 tumor suppressor nonfunctional (26), and most of the others down-modulate the p19Arf protein (18), a protein that leads to p53 activation through effects on Mdm2 (reviewed in reference 24). Consistent with the important role of the p53 pathway in the crisis phase of transformation, pre-B cells from animals lacking functional p53 or the Ink4a/Arf locus that encodes p19Arf bypass crisis (18, 27).

The way in which the p53 pathway is activated in Ab-MLV-transformed cells is not fully understood. Overexpression of Ras and stimulation of Myc have been shown to up-regulate p19Arf in cells stimulated with a variety of oncogenes (6, 24, 35). v-Abl expression activates Ras and stimulates Myc expression, in part through effects on the c-myc promoter (37), providing a route by which p53 could be activated. However, functional Ras and Myc are also required for Ab-MLV-stimulated growth and transformation (22, 23). Other v-Abl-mediated signals also activate pathways that promote growth and survival, including stimulation of JAK and the phosphatidylinositol 3-kinase (PI3-K) pathway (5, 25). Indeed v-Abl-mediated signals transmitted via the PI3-K pathway to Akt may negatively regulate p53 through Akt-mediated effects on the Mdm2 protein (16, 34). Thus, the Ab-MLV-infected pre-B cells that develop into fully established transformants are likely to be those in which growth-stimulatory signals from the v-Abl protein have successfully countered a cellular response that recognizes inappropriate growth and triggers apoptosis. An extension of this idea predicts that at least some Ab-MLV mutants that transform pre-B cells poorly do so as a consequence of their inability to overcome the effects of the p53 response. Consistent with this idea, the poorly transforming Ab-MLV-P90 strain induces disease more rapidly in p53 null mice (36).

Understanding the mechanisms by which weakly transforming Ab-MLV mutants interface with the p53 pathway should shed light on the mechanism by which infected cells develop a fully malignant phenotype. To examine this issue, we tested if an inability to overcome p53-mediated effects is related to the weak transforming phenotype of the P120/R273K Ab-MLV mutant. This mutant encodes a v-Abl protein in which a Lys is substituted for the Arg residue at the base of the phosphotyrosine-binding pocket in the SH2 domain. P120/R273K transforms both NIH 3T3 and pre-B cells poorly, especially at 39.5°C (13). In the present study, infection of bone marrow cells with P120/R273K revealed that the temperature-sensitive phenotype was lost in the absence of p53 or Ink4a/Arf locus products. However, loss of p53 was not sufficient to overcome the defects in growth characteristic of the P120/R273K mutant. These data suggest that v-Abl-mediated signals are affected by a functional p53 pathway at all phases of the transformation process and that a failure to overcome the p53 response may explain the transformation defect of several Ab-MLV transformation mutants.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and viruses. NIH 3T3 cells and 293T cells (7) were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented to contain 10% fetal calf serum (Sigma) and 2 mM L-glutamine (Gibco). Fully established Ab-MLV-transformed pre-B cells were grown in RPMI 1640 medium (Gibco) supplemented to contain 10% fetal calf serum, 2 mM L-glutamine, and 50 µM 2-mercaptoethanol (Sigma). Primary transformants were maintained in the same medium supplemented to contain 20% fetal calf serum. Viral stocks were prepared using Ab-MLV strains in the pMIG vector (11, 28) and the pSV--E-MLV retroviral packaging plasmid (17) as described elsewhere (13, 31). The Ab-MLV-P120 strain and the P120/R273K mutant (13) were used for all infections. Viruses titers were determined by infecting NIH 3T3 cells and analyzing the frequency of green fluorescent protein-positive cells by flow cytometry 24 to 30 h postinfection (30). Equivalent titers of P120 and P120/R273K were used in each experiment. Bone marrow transformation assays were done as described previously using cells from p53 null mice and their wild-type littermates (20, 27). Briefly, 2 x 10⁶ nucleated bone marrow cells were infected and plated in agar medium; the cultures were fed 5 days later, and macroscopic colonies of primary transformants were scored at 10 days postinfection for cultures incubated at 37 or 39.5°C. Primary transformants were removed from agar and plated in liquid medium in 24-well plates; growth and viability were monitored on a daily basis, and when cells filled the well the culture was divided by transferring half of the cells to a fresh well. When the viability exceeded 85% and the cells could be subcultured on a predictable basis, they were transferred to 35-mm-diameter dishes. When the cells could be subcultured routinely and were greater than 90% viable, the cell lines were scored as established (18, 27). The p53 null mice were maintained by mating heterozygous animals originally obtained from a single breeding pair of p53^+/- animals that had been backcrossed to BALB/cJ mice five times and inbred for three generations (Jackson Laboratory). The Ink4a/Arf null mice were originally obtained from R. A. DePinho, backcrossed to C57BL/6 mice for seven generations, and then maintained by brother-sister mating.

Protein analyses. Cell lysates were prepared as described previously (4). Briefly, the cells were washed twice with phosphate-buffered saline (PBS), and the cell pellets were treated with lysis buffer (10 mM Tris [pH 7.4], 1% sodium dodecyl sulfate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The lysates were boiled immediately and sheared through a 25-gauge needle. The amount of protein in each lysate was quantitated using a bicinchoninic acid protein assay kit (Pierce), and 50 µg of each sample was fractionated through an sodium dodecyl sulfate-10% polyacrylamide gel. The proteins were electrotransferred to polyvinylidene difluoride membranes (U.S. Biochemicals) that were blocked with PBS containing 0.2% I-block (Tropix) and 0.1% Tween 20 for at least 1 h. Blotting was performed according to the Western-Light kit protocol (Tropix), utilizing alkaline phosphatase-conjugated secondary antibodies with a CSPD substrate (Tropix). Blots were exposed to Kodak XAR-5 film and subsequently stripped by incubating in a pH 2.2 solution containing 0.2 M glycine and 1% Tween 20 for 3 h at 80°C. After stripping, blots were washed with PBS containing 0.1% Tween 20 and treated with blocking solution prior to reprobing. Proteins were analyzed using anti-ß-actin (Sigma), anti-Myc (Upstate Biotechnology), anti-p19Arf (Novus), and alkaline phosphatase-conjugated anti-mouse immunoglobulin G (Promega) antibodies.

Growth and apoptosis assays. To monitor apoptosis, cells were stained with propidium iodide or merocyanin 540, a dye that specifically stains apoptotic cells (19), and analyzed by flow cytometry; comparable results were obtained with both methods. Growth analysis was carried out by counting trypan blue-stained cells, using a hemocytometer. Duplicate cultures were analyzed at each time point. Population doublings during crisis were evaluated based on the number of times populations were subcultured at a 1-to-2 split ratio. To monitor sensitivity to rapid apoptosis following irradiation, cells were treated with approximately 1,000 rad of -irradiation by using a cesium 137 Gammacell-100 source. The cells were incubated, and the frequency of apoptotic cells was evaluated 12 to 16 h later (12, 26).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The absence of p53 complements the temperature-sensitive phenotype of the P120/R273K mutant. The P120/R273K mutant transforms fewer cells than P120 at 34 or 37°C and is markedly deficient in pre-B-cell transformation at 39.5°C, a temperature typically used for analysis of temperature-sensitive retrovirus transformation mutants (13). To determine if the presence of a functional p53 pathway affects transformation by P120/R273K, bone marrow cells from p53 null mice and wild-type mice were infected with P120/R273K or P120 wild-type virus and the formation of primary transformants was monitored at all three temperatures. As expected (9, 27), P120 induced primary transformants in both wild-type and p53 null cells at all temperatures (Table 1). Although a higher frequency of colonies was observed in the p53^-/- samples, analyses of large numbers of p53^-/- mice indicate that this bone marrow does not always contain a higher frequency of Ab-MLV target cells (27).

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TABLE 1. The absence of p53 partially complements P120/R273K transformationa

Consistent with previous work (13), P120/R273K induced three- to sixfold fewer primary transformants than P120 when wild-type cells were plated at 34 and 37°C and very few transformants when the cells were plated at 39.5°C. These rare colonies were extremely small and barely visible, suggesting that the cells within them grew very poorly. In contrast, when p53 null cells were infected with P120/R273K, primary transformants were observed at similar frequencies at all temperatures. Although the frequency of P120/R273K transformants did not approach that observed with the P120 strain, the ratio of primary transformants induced by P120 and P120/R273K in the p53^-/- samples was similar to that observed for wild-type cells plated at 34 or 37°C. Thus, the absence of p53 complements the temperature-sensitive phenotype of the P120/R273K mutant but does not restore full transformation potential.

Loss of Ink4a/Arf locus products complements P120/R237K transformation at 39.5°C. Previously documented effects of p53 on Ab-MLV transformation require the presence of the p19Arf protein, a molecule that regulates p53 in response to oncogenic signals (24). To determine if this pathway is also involved in temperature-dependent transformation by P120/R273K, bone marrow cells from Ink4a/Arf null mice were infected with P120 or P120/R273K and plated in agar at 37 or 39.5°C, and primary transformants were scored 10 days later. Both P120 and P120/R273K gave rise to primary transformants at both temperatures (Table 2). In addition, similar to results obtained for p53 null animals, the ratio of transformants obtained with P120 and P120/R273K was similar. These data demonstrate that the effects of p53 on primary transformation by P120/R273K involve the products of the Ink4a/Arf locus. Because p19Arf plays the central role in this response when wild-type virus is used (18), it is likely to be the product involved here as well. These data, coupled with those from the p53 null mice, suggest that the p19Arf-p53 pathway can influence primary transformation.

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TABLE 2. The absence of Ink4a/Arf locus products partially complements P120/R273K transformationa

Primary transformants derived using P120/R273K can establish at the nonpermissive temperature in the absence of p53. The second stage of Ab-MLV transformation involves a period when primary transformants display erratic growth and high levels of apoptosis and many primary transformants die (12, 27); cells from p53 null mice bypass the crisis phase of the transformation process (27). To determine if primary transformants from p53 null mice initiated with P120/R273K display a different profile than those expressing P120, cells were explanted from agar and expanded. The viability of all of the cells derived at 37 and 39.5°C remained high, and all of the cells grew well. Most of the primary transformants initiated with P120/273K required several additional days to fill the culture vessel and be considered established (Table 3). At 34°C, all of the P120-initiated primary transformants became established, as did the majority of those derived with P120/R273K. The three colonies that failed to expand in liquid medium contained very few cells, a feature that may have impaired their ability to grow. These data indicate that the absence of p53 allows primary transformants initiated with P120/R273K to establish at the nonpermissive temperature. Thus, the absence of p53 complements the temperature-sensitive phenotype of P120/R273K transformants at both the primary transformant and crisis phases of the transformation process.

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TABLE 3. The absence of p53 complements P120/R273K during establishmenta

The absence of p53 does not restore wild-type growth to P120/R273K transformants. Although p53 complements the temperature-sensitive defect characteristic of the P120/R273K strain, it does not restore transformation potential to wild-type levels, even at the permissive temperature, and primary transformants require somewhat longer to grow out. Wild-type cells transformed with P120/R273K at 34°C grow more slowly than those transformed with P120 (13), suggesting that the P120/R273K v-Abl protein delivers a weak signal for cell growth. To test the possibility that v-Abl-stimulated growth is still compromised in p53 null cells expressing the mutant, the growth of cell lines prepared at 34 and 39.5°C was compared. A total of eight independent clones were compared per temperature, four derived with each virus, including the representatives shown (Fig. 1). In all cases, cells derived by using P120 grew more rapidly than those derived with P120/R273K. These data indicate that the absence of p53 does not compensate for defective growth stimulation by P120/R273K and emphasize that this mutant displays two types of defects, only one of which is compensated by the absence of p53.

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FIG. 1. The absence of p53 does not complement the growth defect of transformants expressing P120/R273K. Transformants generated from p53 null mice at 34 and 39.5°C with P120 and P120/R273K were plated at 2.5 x 105 cells per ml in duplicate, and growth and viability were monitored by counting trypan blue-stained samples at regular intervals. Cells expressing P120/R273K are represented by the circles, and cells expressing P120 are represented by the squares. Each line represents an independent cell line. The experiment shown is representative of at least two independent experiments with these cells and of experiments performed with four additional primary transformants derived using either P120 or P120/R273K. Cell numbers in duplicate cultures varied by <10%.

Crisis is altered in primary transformants expressing P120/R273K derived from wild-type cells. Although analysis of p53 null transformants allows study of cells expressing P120/R273K at the nonpermissive temperature, this approach does not allow study of the impact of the mutant on the crisis phase of transformation, because p53 null transformants bypass this part of the process. To analyze this phase of the transformation process, primary transformants from wild-type mice were studied. A small number of primary transformants are recovered at 39.5°C following infection of wild-type cells with P120/R273K. To determine if these transformants could expand, they were compared to those initiated with P120. Although incubation at 39.5°C did not affect the frequency of P120-infected primary transformants that established, none of the five P120/R273K primary transformants from wild-type cells recovered at 39.5°C survived (Table 3). Thus, even though P120/R273K can mediate some expansion at 39.5°C, the growth of these cells cannot be sustained at this temperature.

In contrast to results obtained at 39.5°C, comparison of primary transformants generated at 37°C revealed that those generated with either virus established at similar frequencies (Table 3). Although very few primary transformants could be established at 34°C, a feature that likely reflects the slow growth of cells at this temperature (3), P120- and P120/R273K-infected cells also appeared to have similar chances of establishing at 34°C. Thus, even though no primary transformants initiated with P120/R273K expanded at 39.5°C, the data from experiments conducted at the other two temperatures indicate that the non-temperature-sensitive defect in P120/R273K does not affect the chance that a primary transformant will establish. Thus, in both wild-type and p53 null cells, this mutation exerts its principal non-temperature-sensitive defect effects at the stage of primary transformation.

Levels of c-Myc and p19Arf are similar in primary transformants expressing P120 and P120/R273K. P120/R273K does not stimulate expression from the v-Abl-responsive element in the c-myc promoter as well as P120 (37), and c-Myc is required for Ab-MLV transformation (22). In addition, expression of p19Arf, a protein required for p53-dependent crisis in wild-type transformants (18), is c-Myc responsive (24). Thus, lower levels of c-Myc or higher levels of p19Arf might influence the frequency of primary transformation or initial growth and survival after explantation when wild-type cells are used for infection. To test this possibility, primary transformants were analyzed by using Western blotting and anti-Myc and anti-p19Arf antibodies. Comparison of cells expressing P120 or P120/R273K revealed that, although levels of p19Arf varied from sample to sample, no consistent difference that correlated with the virus used to infect the cells was evident (Fig. 2), and the pattern of Myc expression was similar to that observed in other Ab-MLV-transformed pre-B cells (13). These data indicate that differences in the levels of c-Myc or p19Arf do not influence the transformation process, at least at 37°C. In addition, these results suggest that v-Abl expression must regulate c-Myc levels via several mechanisms, only one of which involves effects on the v-Abl-responsive element in the c-myc promoter.

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FIG. 2. Myc and p19Arf expression is similar in primary transformants expressing P120 and P120/R273K. Primary transformants from wild-type bone marrow expressing P120 or P120/R273K were plated in liquid medium and lysed at the stage of primary transformation immediately after explant from agar (A) or 1 day later (B). The lysates were analyzed by using Western blotting and anti-p19Arf and anti-Myc antibodies. Each lane represents an independent sample. Controls included fully established Ab-MLV-transformed cell lines that express abundant p19Arf (+) and a fully transformed Ab-MLV cell line derived from an Ink4a/Arf null mouse (-) (18).

P120/R273K wild-type transformants require a longer time to establish than those generated with P120. An important aspect of crisis is the time required for cells to become established. Although the p53 null, P120/R273K transformants expanded slowly, the time required for wild-type cells to pass through this phase is determined by both growth parameters and the duration of the apoptotic response, a feature that is not observed in p53 null cells. For the viruses used here, this parameter can be best compared at 37°C, where a sufficient frequency of cells infected with both viruses can be expanded. Comparison of the kinetics with which primary transformants established indicated that most wild-type cells expressing P120/R273K required almost 2 weeks longer to establish than cells expressing P120 (Fig. 3). The longer crisis period experienced by cells expressing P120/R273K did not reflect differences in the frequency of apoptotic cells (Fig. 4A), suggesting that P120 and P120/R273K v-Abl proteins have a similar impact on this parameter, at least at 37°C. In contrast, calculation of the number of population doublings that occurred during crisis indicated that, on average, primary transformants initiated with both viruses required about the same number of population doublings to pass through the crisis phase (Fig. 4B). These data highlight the inability of the P120/R273K v-Abl protein to stimulate growth as effectively as the v-Abl protein encoded by P120 and suggest that the length of the crisis period is at least partially influenced by the number of doublings a cell population undergoes.

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FIG. 3. P120/R273K primary transformants require longer to establish. Primary transformants were prepared by infecting bone marrow from wild-type mice with either P120 or P120/R273K at 37°C, and the cells were removed from agar and plated in liquid medium (day 0). Growth and viability were monitored, and the cultures were considered established when the cells divided predictably and displayed low levels of apoptosis (12, 18, 27). Each point represents a single culture. The squares represent P120-infected cells; the circles represent P120/R273K-infected cells.

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FIG. 4. Differences in growth rate, but not apoptosis, influence crisis duration. (A) The average number of apoptotic cells in primary transformants was monitored by flow cytometry. The data shown represent analyses of 10 independent P120-infected primary transformants and 7 independent P120/R273K transformants. Similar results were obtained with samples analyzed at two other time points during crisis. (B) The average number of population doublings required for cells to transit crisis was calculated. The calculations shown represent analysis of growth information for all of the primary transformants illustrated in Fig. 2. The error bars indicate standard errors of the means.

P120 and P120/R273K transformants resolve crisis in a similar fashion. Differences in crisis duration could indicate that recovery involves distinct sets of events in cells expressing P120/R273K. For wild-type cells expressing P120, crisis is characterized by the production of high levels of p19Arf in most cell lines. To determine if a similar pattern of p19Arf expression was observed in P120/R273K cells undergoing crisis, samples were examined at several time points during crisis (Fig. 5A). Although individual clones infected with P120 and P120/R273K expressed different levels of p19Arf, as expected (18), no consistent difference in levels that correlated to the virus used to infect the cells was evident, indicating that the two types of cells did not vary with respect to this aspect of crisis.

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FIG. 5. Patterns of p53 mutation and p19Arf expression are similar during crisis and in fully established P120- and P120/R273K-derived transformants. (A) Cells undergoing crisis were lysed and analyzed by Western blotting using anti-p19Arf and anti-ß-actin antibodies. The days indicate the time after explant from agar; each lane represents an independent sample. (B) Fully transformed cell lines were analyzed by using Western blotting and anti-p19Arf and anti-ß-actin antibodies. The samples shown are representative of the cell lines described in Table 4. M, mutant p53; W, wild-type p53. In both panels, controls included a fully established Ab-MLV-transformed cell line that expresses mutant p53 and abundant p19Arf (+) and a fully transformed Ab-MLV cell line derived from an Ink4a/Arf null mouse (-).

Escape from crisis usually involves acquisition of a p53 mutation or loss of p19Arf expression (12, 18, 27). To determine if primary transformants generated from wild-type cells with P120 and P120/R273K acquire p53 mutations during the crisis phase at similar frequencies, fully established transformants prepared at 37°C were screened by using -irradiation. Ab-MLV-transformed cells with functional p53 undergo rapid apoptosis following -irradiation, while cells carrying a p53 mutation remain intact for several days after treatment (12, 18, 26). Screening of these transformants revealed that established cell lines expressing either virus had similar frequencies of p53 mutation (Table 4). Consistent with this pattern, comparison of established P120/R273K and P120 transformants for p19Arf expression by using Western blotting revealed a similar pattern (Fig. 5B). As expected (18), cells that expressed nonfunctional p53 expressed abundant p19Arf, while those that retained a wild-type p53 demonstrated the characteristic low levels of p19Arf. These data suggest that the ways both P120/R273K- and P120-induced primary transformants circumvent crisis are similar.

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TABLE 4. P120 and P120/R273K established cells have a similar frequency of p53 mutationa

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our experiments showing that the absence of p53 or Ink4a/Arf locus products can complement the temperature-sensitive phenotype of the P120/R273K mutant reveal a role for these tumor suppressors at the earliest stage of the Ab-MLV transformation process. Both p53 and p19Arf have previously been implicated in the apoptotic response that characterizes crisis (18, 27). Levels of p19Arf increase as the crisis response escalates (18), and this increased expression is thought to lead to p53 activation by blocking the Mdm2 protein, a negative regulator of p53 (24). However, as shown here, p19Arf can be detected in primary transformants derived from wild-type mice, even though levels are generally lower than they are at later times during the transformation process. Because wild-type cells transformed with P120/R273K could not be recovered at 39.5°C, the possibility that p19Arf levels are higher in such cells could not be tested. Alternatively, the levels of p19Arf observed at 37°C may be sufficient to trigger a p53 response that is capable of overriding the defective stimulatory signals delivered by P120/R273K at 39.5°C. These levels may also be sufficient to affect crisis at 37°C.

Levels of p19Arf expression are similar in primary transformants derived with P120 and P120/R273K, suggesting that a fully functional v-Abl protein can override the effects of the cellular response orchestrated by the p19Arf-p53 pathway during primary transformation. Consistent with this idea, our earlier studies indicated that p53 null bone marrow and bone marrow from wild-type littermates contained about the same frequency of cells susceptible to Ab-MLV (27). Although the number of primary transformants recovered from the p53 null mice examined in this study was somewhat higher than that in control mice, two- or threefold differences in the frequency of primary transformants can be observed among normal, genetically identical mice (12, 27). Interestingly, an independent study using p53 null mice on a different genetic background (36) documented a 10-fold increase in primary transformation when p53 null cells were infected with wild-type virus. This apparent discrepancy may reflect the presence of as-yet-unknown background genes that affect transformation frequency (20). That other study (36) also showed that the absence of p53 did not facilitate transformation by the P90 transformation-deficient mutant. These data suggest that the weakly transforming phenotype of some Ab-MLV mutants does not involve the p53 pathway. Further analyses of these and other Ab-MLV mutants in different genetic backgrounds may help to uncover pathways involved in the p53 response.

Primary transformation is characterized by the proliferation of Ab-MLV-infected cells and is directly dependent on v-Abl-stimulated growth and suppression of apoptosis. Expression of a functional p19Arf-p53 pathway has been most directly associated with apoptosis induction in Ab-MLV-transformed pre-B cells (18, 27). However, expression of P120/R273K in pre-B cells fully transformed by the temperature-sensitive Ab-MLV-P70/H-590 mutant blocks the normal apoptotic response at the nonpermissive temperature but fails to stimulate growth (8, 13; L. Gong, I. Unnikrishnan, K. Parmar, and N. Rosenberg, submitted for publication). These data and the reduced ability of P120/R273K transformants derived from wild-type mice to grow at the nonpermissive temperature (13) suggest that p53 suppresses the growth of the cells. The pathways by which this might be accomplished require further study. However, p53 is known to alter cell cycle progression through effects on p21Cip1, cyclin G, GADD45, and other proteins (29). Primary transformants from p21Cip1 null mice undergo crisis similar to those derived from wild-type mice (27), suggesting that this protein may not be the target. However, deficient growth stimulation by the P120/R273K protein may allow this or another p53-regulated cell cycle progression protein to assume a larger role in determining the outcome of transformation.

The deficiencies in growth stimulation characteristic of P120/R273K are reinforced by the analysis of crisis in primary transformants prepared using wild-type cells and P120/R273K. At 37°C, the slower growth of P120/R273K-expressing cells extends the crisis period. However, cells expressing P120 and P120/R273K require about the same number of population doublings to escape from crisis. In addition, similar to P120 transformants (18), recovery is correlated with inactivation of the p19Arf-p53 response, a change mediated by either p53 mutation or p19Arf down-regulation. A correlation with population doublings seems likely, because the time required to become established would be influenced by the chance that such a change will occur and be fixed in the population. The observation that p53 mutations, escape from crisis, and establishment occur with accelerated kinetics in the absence of DNA mismatch repair (12) is consistent with this idea. Because similar frequencies of wild-type primary transformants expressing P120 and P120/R273K become established and display similar frequencies of p53 mutation, the defect in P120/R273K does not appear to affect the chances that such a mutation will occur.

Even though the absence of p53 or Ink4a/Arf locus products complements the temperature-sensitive defect displayed by the P120/R273K mutant, full transformation potential is not restored, even at 34°C. The mutation in this strain substitutes a Lys for the ßB5 Arg residue of the SH2 domain; this Arg residue plays a dominant role in binding phosphorylated tyrosine residues, and mutations affecting the isolated domain reduce the binding of phosphorylated peptides dramatically (1, 2, 15). The formation of signaling complexes mediated by the SH2 domain is critical for v-Abl-transmitted signals, and v-Abl proteins that lack an SH2 domain have very limited transformation potential (14). The reduced ability of the P120/R273K v-Abl protein to assemble critical signaling complexes probably contributes to the overall transformation defect displayed by the mutant.

Defects in signaling to Ras and in activating the v-Abl-responsive element in the c-myc promoter have been documented for the P120/R273K v-Abl protein (13, 37), and both the Ras and Myc pathways are required for Ab-MLV-mediated transformation (22, 23). Interestingly, although P120/R273K primary transformants prepared at 37°C express c-Myc at levels similar to those observed in primary transformants initiated with P120, expression of P120/R273K is not sufficient to sustain c-Myc expression at 39.5°C in pre-B cells transformed by Ab-MLV/P70-H590 (13). When considered together, these data suggest that elevated temperature alters SH2-mediated interactions and that the temperature-sensitive phenotype of P120/R273K reflects temperature-dependent interactions with some signaling complexes. In contrast, the defect in transformation that is observed at the permissive temperature probably reflects a failure to form other complexes at any incubation temperature. The realization that interactions which are important for temperature-dependent transformation interface in some fashion with the p53 pathway may help to identify these intermediates.

 
This work was supported by a fellowship from the Leukemia and Lymphoma Society of America to I.U. and grants CA 33771 and CA 24220 from the National Cancer Institute.

 
* Corresponding author. Mailing address: Jaharis 808, Tufts Medical School, 150 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2143. Fax: (617) 636-0337. E-mail: naomi.rosenberg{at}tufts.edu.

Present address: Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, June 2003, p. 6208-6215, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6208-6215.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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