Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia


The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms β and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.


The tumor-suppressor p53 is a transcription factor that integrates cell cycle checkpoint cues and DNA damage signals and initiates a transcriptional program that in normal cells induces cell cycle arrest or apoptosis on cell stress (Hainaut, 1995; Vousden and Lane, 2007; Levine and Oren, 2009). Wild-type p53 is usually required to sensitize cancers to chemotherapy, subsequently; cancers with TP53 mutations often are chemoresistant (Aas et al., 1996; Levine, 1997; Preudhomme and Fenaux, 1997; Petitjean et al., 2007). Recent mapping of the structure of the p53 gene family (including p63 and p73) revealed employment of different promoters and alternative splicing of transcripts, resulting in several isoforms of the p53 protein with tissue-specific expression (Bourdon et al., 2005; Rohaly et al., 2005). The p53 isoforms have been suggested to exhibit different functions and target genes, although their exact role remains elusive (Chen et al., 2009; Fujita et al., 2009; Graupner et al., 2009). Furthermore, the nature of p53 splice form regulation and the clinical significance of p53 isoform expression in cancer are not understood. For this reason, we investigated the relationship between p53 isoforms present in patient materials and clinical outcome.

Acute myeloid leukemia (AML) is a rapidly developing hematological cancer of the bone marrow with a low frequency of p53 mutations (Tallman et al., 2005; Estey and Dohner, 2006; Haferlach et al., 2008). Estimation of prognosis, that is, risk of later leukemia relapse, for patients with AML is typically based on the presence of recurrent cytogenetic aberrations (Grimwade et al., 1998; Löwenberg et al., 1999). Further important prognostic markers include absences of AML cells in the bone marrow after first chemotherapy cycle (Mayer et al., 1994; Wheatley et al., 1999), and mutational analysis of the molecular chaperone nucleophosmin (NPM1) (Falini et al., 2005) and the receptor tyrosine kinase FLT3 (Kottaridis et al., 2001; Löwenberg, 2008). Although mutation of NPM1 is associated with good prognosis (Dohner et al., 2005; Schnittger et al., 2005; Verhaak et al., 2005), mutations in the juxtamembranous region of FLT3 internal tandem duplication (FLT3-ITD) have been found to be a particularly strong and independent predictor of disease relapse after chemotherapy (Gale et al., 2005b).

We have previously shown that the level of signaling through phosphoprotein networks correlates with patient responsiveness to chemotherapy (Irish et al., 2004). Several signaling pathways converge on p53 (Kojima et al., 2007), and we recently reported p53 hyperphosphorylation in sub-populations of AML patients indicating that p53 function may be affected in AML although patients do not display p53 mutations (Irish et al., 2007). We have previously detected mRNA from p53 isoforms β and γ in AML patient cells and the corresponding protein appeared detectable by two-dimensional immunoblot (Anensen et al., 2006). p53 isoform β is suggested a functional role in colon adenoma but not in colon cancer, and isoform γ expression may be related to better prognosis in TP53 mutated breast cancer (Fujita et al., 2009; Bourdon et al., 2011). Consequently, we asked whether regulation of wild-type p53 protein isoforms in AML could be coupled to patient outcome. We visualized individual AML patient p53 biosignatures through protein isoform separation and detection by two-dimensional gel electrophoresis and immunoblot (2DI) (Anensen et al., 2006; Irish et al., 2007). mRNA of Δ133 p53β and Δ133 p53γ isoforms were not detected in AML patients examined, while all patients tested expressed mRNA of p53 full-length, p53β and p53γ isoforms. Using image analysis and a highly validated automated correlation algorithm (Van Belle et al., 2006), we coupled p53 isoform expression to overall survival, chemotherapy-resistant disease and FLT3 and NPM1 mutational status. Our results show that protein expression of the full-length p53 isoform predicts adverse prognosis while expression of p53 isoforms β and γ is associated with long-term survival. In vitro anthracycline chemotherapy in osteosarcoma cell line SAOS-2 expressing increased p53 isoforms β and γ relative to full-length p53 demonstrated elevated chemosensitivity. This indicates that the protein biosignature of p53 isoforms in the absence of any mutations may convey information on chemoresistance in cancer.


p53 protein biosignatures and analysis

Two-dimensional immunoblotting (2DI) with antibody detection of the amino-terminal region of the p53 protein in AML patients expressing wild-type p53, revealed a reproducible pattern of two main isoforms with different molecular weight, as well as several alternatively charged variants. Furthermore, various patients clearly displayed distinct p53 biosignatures (Figure 1a).

Figure 1

Detection of a p53 protein biosignature. (a) Shows p53 protein detected in protein extract from AML patient cells (P18, P19 and P48) after two-dimensional immunoblots using an antibody with a N-terminal epitope (bp53-12; epitope amino acid residues 20–25). Two distinct regions of protein expression were revealed. In order to elucidate the nature of these expression patterns, the p53 null cell line SAOS-2 was transfected with constructs encoding known p53 isoforms. (b) Shows that the observed isoforms were the full-length p53 protein, p53β and p53γ (detected together in the same region). Transfection experiment was performed in three independent replicates.

To identify the components of the p53 biosignature, we transfected the p53−/− cell line SAOS-2 with complementary DNA constructs representing p53 full-length and p53β and p53γ isoforms. This identified the lower mobility isoforms (53 kDa) as p53 full-length and the higher mobility isoforms (46 kDa) as a combination of p53β and γ (Figure 1b). p53 isoform mRNA expression was confirmed by reverse transcriptase–PCR and identified p53 full-length, β and γ mRNA in all patients examined (see Table 1) (Bourdon et al., 2005).

Table 1 Overview over acute myeloid leukemia patients included in this study

The p53 biosignatures obtained by 2DI analysis of different AML patient samples were highly heterogeneous (Figure 1a). In order to explore whether p53 isoform expression in different patients reflects disease progression, we developed a method to align 2DI images of p53 biosignatures and correlate presence of p53 isoforms and isoelectric variants with clinical data. Thus, we combined 2DI image analysis and an automated Spearman rank-order correlation test exploring pixel-by-pixel biomarker data and biological parameters as previously described (Van Belle et al., 2006). Using this method, we correlated p53 isoform expression with clinical outcome and mutational status of FLT3 and NPM1 to investigate the significance of p53 biosignature variations (Figure 2).

Figure 2

Correlation analysis of p53 protein by two-dimensional immunoblot and clinical parameters in AML patients. In the two-dimensional immunoblot (2DI), the proteins are dispersed in two dimensions, by isoelectric point and molecular size (a). 2DI image correlation relies on an aligned, normalized stack of 2DI images and a numerical label associated with every gel (b). Pixel-by-pixel correlation between gel intensities and the external variable creates a new image, showing areas in the gel that relate to the external parameter (c). The correlation value is reflected in the coloring of the patterns. Green color indicates no correlation while coloring toward the red and blue end of the spectra indicate correlation and inverse correlation, respectively. The patient samples were analyzed by 2D1 twice, and with few exceptions, each patient was represented with two images in the correlations.

Correlation of p53 biosignature with molecular prognostic markers

First, we examined whether individual patient p53 biosignatures correlated with molecular markers for prognosis. Cells from all patients involved in the study were subjected to p53 2DI analysis (separate for patient set 1 and 2), image data processed and the presence or absence of p53 full-length and p53β/γ correlated with clinical data (Figure 2). AML patient materials where collected consecutively at the time of diagnosis and processed for immunoblot imaging in two separate rounds. The two data sets were analyzed separately (n=38, n=30) and combined, revealing similar results (Figure 3a, Supplementary Figure S1). Clinical and biomarker data on both patient populations are presented in Table 1, and can be summarized as follows: patient set 1, n=38, average age 61 years, M/F=20/18 patients, FLT3 wt/ITD=20/16, NPM1 wt/mutated=21/7; patient set 2, n=30, average age 61.2 years, M/F=18/12, FLT3 wt/ITD=12/10, NPM1 wt/mutated=15/4. For patients under 60 years of age, treatment was initiated with an induction cycle consisting of idarubicin and cytarabine (Löwenberg et al., 2003), to be followed by four cycles with high dose (2000 mg/m2) cytarabine (Mayer et al., 1994). In contrast, patients above 60 years of age received an induction cycle consisting of daunorubicin and cytarabine (Mayer et al., 1994), followed by three cycles of chemotherapy consisting of cytarabine at a lower dose (200–400 mg/m2), mitoxanthrone, amsacarine and etoposide (Wahlin et al., 1997). Elderly AML patients medically unfit for chemotherapy was treated with best supportive care (Table 1, ‘resistant disease’ indicated by ‘n.d.’)

Figure 3

Mutational status of positive prognostic AML disease marker NPM1 in relation to p53 protein biosignature. Status for mutation of NPM1 was determined at diagnosis and (a) shows presence or absence of NPM1 mutation in correlation with p53 biosignature in the two patient sets (n=32 and n=18, correlation coefficients 0.53 and 0.60, respectively, t=4.05; t=4.37; P=0.00021; P=0.00011) (p53 full-length is indicated by ‘FL’ and p53β/γ is indicated by ‘β/γ‘). (b) Shows the two image sets combined (n=50, correlation coefficient 0.4, t=3.85, P-value=0.00024). The two sets have highly similar correlation images including the maximum correlation values. Increasing intensity of red color indicates presence of NPM1 mutation. A positive correlation with the p53β/γ region was detected. No sub-p53β/γ region was observed to correlate to NPM1 mutation, exemplified by one patient known to have NPM1 mutation (P16; Table 1).

Mutations in the molecular chaperone NPM1 have been linked to a favorable prognosis in AML with the present induction treatment containing anthracycline and cytarabine (Falini et al., 2005). Thus, NPM1 mutations would theoretically be expected to occur in cancers expressing the p53 biosignature associated with prolonged survival. We observed a positive correlation between distinct p53β/γ spots with NPM1 mutation both in patient set 1 and 2. (Figure 3a; positive correlation evident is yellow to red colors). A combined correlation analysis of all 47 patients with known NPM1 mutation status confirmed the results within sets 1 and 2 (correlation coefficient 0.4, t=3.85, P-value=0.00024) (Figure 3b), see example of representative patient (P16, Figure 3b, rightmost panel; survival >64 months; alive May 2007). It was evident that all (11/11) of NPM1 mutated patients expresses predominantly p53β/γ.

Oncogenic mutations in receptor tyrosine kinases are a common feature in a wide range of malignancies. In AML, an insertion (ITD) in the juxtamembrane region of receptor tyrosine kinase Flt3 is the single strongest marker for disease relapse (Gale et al., 2005a); thus, we examined this duplication across the complete patient material (Table 1). The patient data on FLT3-ITD were coupled to the compiled p53 biosignature data and a new correlation analysis performed. As evident from Figure 4a, the independently analyzed biomarker FLT3-ITD positively correlated with p53 full-length in both set of AML patients (correlation coefficient 0.61, t=4.75, P=0.00024 and 0.52, t=3.90, P=0.0004; yellow to red color). This is consistent with the analysis of overall survival and p53 isoform expression, where the p53 full-length region is negatively associated with long survival (Figure 5a). These data suggest that the biosignatures based on correlation with FLT3-ITD or survival, respectively demonstrates clear similarities. In the p53β/γ region, FLT3-ITD correlation analysis discerned two distinct populations of p53 protein with slightly different mobility. Presence of FLT3-ITD was inversely correlated with the existence of a p53β/γ sub-population of higher molecular weight (−0.51, t=−3.65, P=0.00076; appears in blue color) and positively correlated with a different p53β/γ sub-population with somewhat lower molecular weight (correlation coefficient 0.45, t=3.11, P=0.0035) (Figure 4a, leftmost panel; appears yellow). When reexamining the individual patient data, this split p53β/γ region could be identified in 2DI images of patients carrying FLT3-ITD (exemplified by patient P47, Figure 4b, right and insert) but not in patients with FLT3 wild-type, suggesting that this is a specific p53 expression pattern associated with the presence of FLT3-ITD. Interestingly, no sub-p53β/γ populations were identified in the NPM1 analysis.

Figure 4

Mutational status of negative prognostic disease marker FLT3-ITD in relation to p53 protein biosignature. Status for mutation in the juxtamembrane region of Flt3 was also determined at diagnosis. (a, b) shows presence or absence of FLT3 length mutation (FLT3-ITD) in correlation with p53 biosignatures (n=29, n=22 and n=61) (p53 full-length is indicated by ‘FL’ and p53β/γ is indicated by ‘β/γ’). Increasing intensity of red color indicates presence of FLT3-ITD, and increasing intensity of blue color indicates wild-type gene for FLT3. Presence of FLT3-ITD corresponded with expression of the full-length p53 protein (FL). Presence of FLT3-ITD also corresponded to presence of a sub- p53β/γ region as exemplified by one patient known to be positive for FLT3-ITD (P47; Table 1) (as shown in panel (a), right and inset).

Figure 5

p53 protein biosignatures in AML at time of diagnosis and their correlation with survival after chemotherapy and disease resistant to induction chemotherapy. (a) Shows the p53 protein biosignatures of treated patients (n=36) correlated to resistant disease (correlation coefficient −0.56, t=−5.01, P=0.000006) (Supplementary Figure S1a shows correlation of the two patient sets separately) (p53 full-length is indicated by ‘FL’ and p53β/γ is indicated by ‘β/γ’). Increasing intensity of blue color indicates therapy success and induction of remission. Expression of the p53β/γ isoforms was found to inversely correlate with chemoresistance and thus indicate response to therapy if expressed. (b) Shows the p53 protein biosignatures of all patients (n=68) correlated to months of survival after start of treatment. Increasing intensity of red color indicates long survival, and increasing intensity of blue color indicates short survival. Expression of the p53β/γ isoforms was found to correlate with increased survival. The full-length protein was inversely correlated and thus predicts shorter survival (correlation coefficients 0.39 and −0.37, respectively, t=4.36; t=−4.1; P=0.00003; P=00008). (c) Shows specific patients known to have long survival (P21 and P22; Table 1). These patients were shown to coincide with this predicted pattern, which is also resembles the p53 biosignature of peripheral blood mononuclear cells (PBMC) from healthy donors. Survival in months after diagnosis is also illustrated in a Kaplan–Meier plot in Supplementary Figure S2.

Correlation of p53 biosignature with clinical parameters

To assess whether p53 biosignatures provided any correlative information on therapy effect, we next investigated the association between p53 protein expression and response to chemotherapy. Resistance to therapy was defined as >5% AML blasts remaining in the bone marrow 14–21 days after start of the first induction cycle and therapy response data were coupled to the compiled p53 biosignature data set. By correlation analysis, a significant inverse correlation (−0.56, t=−5.01, P=0.000006) of the p53β/γ region was shown to be associated with resistant disease after the first course of chemotherapy (Figure 5a; appears blue). High expression of p53β/γ thus indicates favorable prognosis with response to chemotherapy and long survival (Figures 5a and b) whereas presence of p53 full-length indicates short survival. In contrast, p53 full-length was not informative with respect to resistance to treatment (Figure 5a).

In support of the correlation between p53β/γ and survival, individual patients specifically known to have long overall survival were found to express the p53β/γ pattern. Interestingly, this pattern resembles the biosignature of normal peripheral blood mononuclear cells from healthy donors (Figure 5c). We also tested if patient age correlated with a certain p53 biosignature (Supplementary Figure 1b). Maximum positive correlation (higher age) was 0.36 (t=4.05, P=0.000096) for p53 full-length, and may reflect the lower level of NPM1 mutations found in elderly AML (Verhaak et al., 2005) (age <50 years: 5 of 17 patients with NPM1 mutation; age >70 years: 1 of 27 patients with NPM1 mutation; Table 1) as well as the predominance of complex karyotype (Estey and Dohner, 2006).

Longer patient survival positively correlated with p53β/γ expression and correlated negatively to expression of p53 full-length (correlation coefficients 0.39 and −0.37, t=4.36; t=−4.1; P=0.00003; P=00008, respectively) (Figure 5b). This correlation was also consistent when patient set 1 and 2 were analyzed separately (Supplementary Figure 1a). This clearly demonstrates a connection between survival and response to treatment. Furthermore, the positive correlation of p53β/γ with NPM1 mutation was consistent with the inverse correlation of p53β/γ and resistance to therapy. p53 full-length demonstrated weak (FLT3-ITD) or absent (NPM1 mutation; resistant disease) correlation to prognostic markers, while longer survival had a negative correlation to p53 full-length.

The impact of p53 bioprofile (p53 full-length/β/γ isoforms) on chemosensitivity was tested experimentally in the TP53 null osteosarcoma cell line SAOS-2. SAOS-2 cells were transiently transfected with selected ratios of p53 full-length, p53β and p53γ complementary DNA and treated for 24 h with the anthracycline doxorubicin in vitro before determination of proliferation by 3H-thymidine and nuclear condensation/fragmentation (Figure 6). SAOS-2 cells transfected with p53β and p53γ in excess as relative to the full-length transcript (1:1:1) indicated increased sensitivity to doxorubicin, and this combination displayed more cell death than singular or duplex transfections (data not shown). This indicates experimental support to the hypothesis that AML leukemic cells with a favorable level of β and γ relative to full-length p53 should be the responders after an anthracycline-based therapy regime.

Figure 6

p53 protein isoforms modulate doxorubicin sensitivity. (a) Immunoblot verification of SAOS-2 cells transfected with complementary DNA (cDNA) constructs with p53 full-length, p53β, p53γ or vector control. (b, c) Each 96-well were transfected with a total of 0.1 μg cDNA, consisting of either single plasmids or combinations of plasmids at different ratios. p53 full-length, p53β and p53γ were transfected in combination at three different ratios, 1:1:1 (0.033 μg of each), 2:1:1 (0.05 μg p53 full-length, 0.025 μg p53β and 0.025 μg p53γ) and 18:1:1 (0.09 μg p53, 0.005 μg p53β and 0.005 μg p53γ). At 24 h after transfection, doxorubicin was added at concentrations of 100 nM, 250 and 500 nM and cells were left to incubate for 24 more hours. Cells identically transfected, but not receiving treatment were included as transfection control. (b) [3H]-thymidine was incorporated in the last 18 h of the experiment and DNA subsequently harvested. [3H]-thymidin counts for doxorubicin-treated cells were divided by the [3H]-thymidin counts for the untreated transfection, to give a ratio whereby values>1 indicates increased proliferation and values <1 indicates reduced proliferation rates. Statistical difference between the transfection-ratios were calculated by comparison with the 1:1:1 transfection. Asterisks indicate significant difference to the 1:1:1 transfection with the corresponding doxorubicin treatment *P-value <0.05 and **P-value <0.01. (c) The experiment was stopped by adding 4% formalin containing Hoechst 33342. Normal and non-normal nuclei were counted by three evaluators by blinded counting. Approximately 300 cells were counted in each well, and each condition was examined in three parallels in three separate experiments. Cell death fraction is presented relative to cell death in transfection control. **P-value <0.01 indicate significant difference to the 1:1:1 transfection with the corresponding doxorubicin treatment.


Most cancers comprise a multitude of individualized genetic alterations (Parsons et al., 2008; Shah et al., 2009) and extensive use of cytogenetics and molecular diagnostics in AML has proven high heterogeneity among leukemia patients (Löwenberg, 2008). Genetic changes in AML frequently alter signal transduction either directly or indirectly (Irish et al., 2004), subsequently reflected in posttranslational modifications of protein stress sensors and tumor suppressors like p53 (Irish et al., 2007). Given the complexity of p53 regulation including isoform expression and post-translational modifications, p53 protein is hypothesized to integrate multiple sources of information about intracellular signaling with relevance for chemoresistance (Fridman and Lowe, 2003; Irish et al., 2007).

We used 2DIs with an antibody directed toward an N-terminal epitope (Bp53-12, epitope 20–25) in our p53 biosignature analysis. This N-terminal epitope is present in p53 full-length and p53 splice forms p53β and p53γ This N-terminal epitope comprise residues that is targeted by a number of kinases involved in stress responses and chemoresistance, making this region of the p53 protein attractive for analysis of disease prognosis. The resulting p53 biosignature obtained includes p53 full-length, p53β and p53γ (Figures 1 and 2). Here, we used our novel correlation algorithm (Van Belle et al., 2006) to investigate differences in mRNA splice-directed protein isoform expression.

Mutation of NPM1 is associated with favorable outcome in AML treated with the current chemotherapy including anthracycline and cytarabine (Falini et al., 2005). NPM1 is involved in ribosomal biogenesis and may as such contribute to the regulation of p53 isoform expression (Grisendi et al., 2006). In our correlation analysis, NPM1 mutation was associated with high expression of p53β and p53γ proteins (Figures 3a and b). In contrast, FLT3 mutations were associated with high expression of full-length p53 (Figures 4a and b).

Activation analysis of phosphoprotein signaling networks, including FLT3 signaling, has previously been suggested to predict chemoresistance in AML (Irish et al., 2004). We speculate that signaling from the activated Flt3 kinase may affect p53 function. In some leukemia cell lines, Flt3 activates the p38 and ERK1/2 kinases (Srinivasa and Doshi, 2002; Yeh et al., 2004; Komeno et al., 2005), which directly phosphorylate p53 (Yeh et al., 2004; Komeno et al., 2005). Flt3-mediated activation of downstream signaling pathways may thus stabilize the p53 full-length protein, and the p53 biosignature therefore reveal a high expression of the p53 full-length protein as observed (Figure 4). This also supports our observation that specific FLT3-ITD corresponds to p53 hyperphosphorylation (Irish et al., 2007). On the basis of these previous results, it appears that FLT3-ITD-driven p53 hyperphosphorylation is accompanied by a downstream block of p53 signaling through anti-apoptotic mechanisms (Irish et al., 2007). This situation will allow accumulation of p53 full-length but no subsequent apoptosis, resulting in chemoresistance and adverse prognosis. The relation of FLT3-ITD and p53 is supported by a recent report, where FLT3-ITD is suggested to confer p53-dependent resistance to the anthracycline doxorubicin in a mouse model of AML (Pardee et al., 2011). Interestingly, inhibition of Flt3 signaling seems to reduce the correlation with p53 full-length (data not shown), supporting our observation that these signaling pathways could modulate the p53 biosignature in vivo.

The correlation analysis between p53 expression and FLT3-ITD revealed two distinct populations in the p53β/γ region (Figure 4a). The nature of these two subdomains of p53β/γ isoforms has not been determined. However, the appearance of the additional sub-population could be detected in a majority of patients (15/27) who carry FLT3-ITD and may be a distinct feature of this disease.

Disappearance of AML blasts from the bone marrow is an important clinical parameter in AML treatment, and persistence of AML cells 14 days after initiation of chemotherapy is associated with an increased risk of later AML relapse. Thus, detection of persistent AML cells qualifies for immediate additional therapy (Mayer et al., 1994; Wheatley et al., 1999). Our analysis of chemoresistant disease demonstrated negative correlation with protein expression of the p53β/γ splice variants. As the presence of p53 β/γ was positively correlated with the good prognostic marker NPM1 mutation (Figures 3a and b), we questioned whether the presence or absence of p53 protein isoforms provided information about overall survival (Figures 5a and b). Our data strongly suggest that high expression of p53β and p53γ combined with low expression of p53 full-length correlate with longer patient survival (Figures 5a and b). Cells from healthy donors also demonstrate this specific isoform distribution (Figure 5c) suggesting high p53β and p53γ expression levels may reflect a clinical situation in which the cells may still respond to chemotherapy-induced stress. Our in vitro experiments in SAOS-2 cells support this conclusion, where cells expressing p53β/γ at levels exceeding p53 full-length are the most sensitive to doxorubicin (Figure 6). Furthermore, our observation is consistent with enhanced Bax expression and chemotherapy response reported in cell lines predominantly expressing p53β (Bourdon et al., 2005).

The detected inverse correlation between resistant disease and p53β/γ expression (Figure 5a) supports the suggested positive prognostic impact of p53β/γ expression. These data suggest that the balance between different p53 isoforms could set the threshold for response to chemotherapy. The p53 protein biosignature as such may reflect cancer cell-specific p53 isoform expression and post-translational modifications directed by the molecular perturbations behind the leukemogenesis. This is supported by the observation that several of these perturbations protect against chemotherapy-induced apoptosis. The fact that the pixel-by-pixel correlation technique used in this study does not return a simple value or vector that describe the patient's isoform profile prevent us from performing multivariate regression analysis of the isoforms because of the large number of pixels to be analyzed for each image. On the other hand, it is not likely that the p53 isoform profile is an independent predictor of outcome, because there is experimental and clinical evidence that chemosensitivity depend on p53 status, FLT3-ITD may direct p53-dependent anthracycline resistance, and finally that NPM1 is interacting with the p53 protein (Grisendi et al., 2006).

The p53 pathway is critical in cancer development but how regulation of p53 affects the clinical setting has remained elusive (Hall and McCluggage, 2006; Chrisanthar et al., 2008). Given the central role of p53 in cell signaling networks, the biosignature is likely to be altered depending on the activity of signaling molecules in the cell. This suggests that perturbations in signaling pathways in cancer cells will be reflected at the level of p53 protein expression. We therefore hypothesize that cancer cell p53 biosignatures contain information of relevance to cancer therapy. In addition, decoding p53 protein biosignature may become a useful instrument in the future evaluating treatment response (prediction) as well as prognosis (Lonning, 2003) and may provide a tool for customizing signaling targeted therapy. In a future perspective, it would be interesting to combine the correlation method with more extensive, sensitive and quantitative analysis of p53 isoforms and post-translationally modified versions of the protein, employing methods like targeted mass spectrometry.

Materials and methods

Patients, separation of leukemia cells, mutational and gene deletion analyses

The study was approved by the Ethics Committee at the University of Bergen/Haukeland University Hospital (REK Vest). Samples were collected after informed consent. During the period 1992–2006, we collected peripheral blood blasts from 68 AML patients. The clinical and biological characteristics of the patients are presented in Table 1. Cell separation, cryopreservation, culture and protein sample preparation of patient AML blasts and peripheral blood mononuclear cells from healthy donors were performed as previously described (Gjertsen et al., 2002; Bruserud et al., 2003; Irish et al., 2004). Presence of mRNA for p53 isoforms was examined by reverse transcriptase–PCR in approximately 50% of the patients according to previously described procedure (Bourdon et al., 2005).

Mutational analysis of the TP53 gene in our AML material had previously been performed by temporal temperature gradient gel electrophoresis in 27 of 68 patients as part of another study (Irish et al., 2007). Only two patients displayed TP53 mutations (exon 7 codon 238: G->A; exon 5, codon 165: C->T). Potential deletions of the 11 exons of TP53 were examined by the Multiplex ligand probe amplification analysis, performed according to the manufacturer's instructions (SALSA multiplex ligand probe amplification TP53 kit, P056-A2; MRC-Holland, Amsterdam, The Netherlands). In the patient samples, the peak areas of all multiplex ligand probe amplification products resulting from TP53-specific probes were first normalized by the average of peak areas resulting from control probes specific for locations outside of chromosome 17. A ratio was then calculated where this normalized value was divided by the corresponding value from a sample consisting of pooled DNA from 10 healthy individuals. A sample was scored as having a reduced copy number at a specific location if this ratio was below 0.75, and as having an increased copy number if the ratio was above 1.25. Available experimental data and existing literature therefore suggest that detected p53 protein is wild type in more of 90% of the patients (Fenaux et al., 1992; Schottelius et al., 1994). Cytogenetics and mutational analysis (NPM1 and FLT3-ITD mutations present or absent) was performed as previously described (Irish et al., 2004, 2007).

Sample preparation and immunoblot procedure for p53 protein analysis

Cells for analysis of p53 were lysed in 7% trichloroacetic acid and protein prepared as previously described (Irish et al., 2007). SAOS-2 cell line (DSMZ, The German Resource Centre for Biological Material) was cultured in McCoy's 5A medium (Sigma-Aldrich, St Louis, MO, USA) with 15% fetal calf serum (HyClone, South Logan, UT, USA). Constructs for p53 isoforms (Bourdon et al., 2005) were transfected into the p53 null cell line SAOS-2 using the FuGENE 6 Transfection Reagent (Roche Diagnostics, GMbH, Mannheim, Germany), according to the manufacturer's instructions. After treatment, the cells were left to incubate for 24 h at 37 °C before analysis with one-dimensional gel electrophoresis, according to standard procedures, or two-dimensional gel electrophoresis as previously described (Irish et al., 2007).

p53 protein was detected using primary bp53-12 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), p53β was detected using primary K8.1 antibody (data not shown) (Bourdon et al., 2005) and β-actin was detected by β-actin, sc-47778 (Santa Cruz Biotechnology). Primary antibodies were followed by secondary horseradish peroxidase conjugated mouse antibodies (Jackson ImmunoResearch, West Grove, PA, USA) visualized using the Supersignal West Pico or Supersignal West Femto system (Pierce Biotechnology, Inc., Rockford, IL, USA). Chemiluminescence imaging was performed using a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA).

Correlation of two-dimensional immunoblots with clinical features

The p53 2DI gel images were aligned and protein expression levels were normalized, allowing investigation of expression within different areas of the gel as well as the morphology of the expressed protein spots. After normalization, a Spearman rank-order correlation test was applied (Van Belle et al., 2006). One test was performed for every pixel position in all gels. The protein expression in the same pixel position in all the gels was one parameter, and a clinical feature (survival, resistant disease, FLT3-ITD, NPM1) the other. Depending on the outcome of the test, the pixel position was colored differently (positive correlation, red; negative correlation, blue). This resulted in a correlation image, which could be used to pinpoint the position of protein expression for a specific clinical outcome. The correlation value was used to calculate the t-value (Van Belle et al., 2006) and the probability of significance (P-value) was found by calculating the t-distribution in a two-tailed Student's t-test with n–1 degrees of freedom.

Proliferation assay and determination of cell death in doxorubicin-treated SAOS-2 cells

DNA synthesis was determined by [3H]-thymidine incorporation as previously described (Bruserud et al., 2003). SAOS-2 cells (TP53 null) were seeded in 96-well tissue plates (7.5 × 103 cells per well), and left to settle for 24 h before they were transfected with construct for p53 isoforms (Bourdon et al., 2005) using FuGENE 6 Transfection Reagent. At 24 h after transfection, the cells were treated with doxorubicin (Pfizer Inc., New York, NY, USA) for additional 24 h. [3H]-thymidine (1 mCi per well; TRA310, Amersham International, Amersham, UK) was added in the last 18 h of the experiment. DNA was harvested by liquid scintillation counting (Packard Microplate Scintillation and Luminescence counter, Perkin Elmer Life and Analytical Science, Inc., Waltham, MA, USA).

Cell death was determined by examination of nuclear morphology (Gjertsen et al., 1994). After fixation and staining of transfected and treated cells (as described above) with 4% formaldehyde, 10 μg/μl Hoechst 33342 (Enzo LifE Sciences AG, Lausen, Switzerland), cell number per field, cellular and nuclear morphology were examined under epifluorescent microscopy. Normal and non-normal nuclei were counted by three evaluators by blinded counting. Approximately 300 cells were counted in each well, and each condition were examined in three parallels in three separate experiments. The transfection of SAOS-2 cells was verified by western blotting of cells lysed 48 h after transfection with complementary DNA. Statistical analysis of viability and cell death data were performed using GraphPad PRISM (version 5.0b, GraphPad Software, Inc., La Jolla, CA, USA) software. Paired Student’s t-test was used to compare two and two groups.


  1. Aas T, Borresen AL, Geisler S, Smith-Sorensen B, Johnsen H, Varhaug JE et al. (1996). Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients. Nat Med 2: 811–814.

  2. Anensen N, Oyan AM, Bourdon JC, Kalland KH, Bruserud O, Gjertsen BT . (2006). A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia. Clin Cancer Res 12: 3985–3992.

  3. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP et al. (2005). p53 isoforms can regulate p53 transcriptional activity. Genes Dev 19: 2122–2137.

  4. Bourdon JC, Khoury MP, Diot A, Baker L, Fernandes K, Aoubala M et al. (2011). p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients. Breast Cancer Res 13: R7.

  5. Bruserud O, Hovland R, Wergeland L, Huang TS, Gjertsen BT . (2003). Flt3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt3-ligand effects in AML cell populations with and without genetic Flt3 abnormalities. Haematologica 88: 416–428.

  6. Chen J, Ng SM, Chang C, Zhang Z, Bourdon JC, Lane DP et al. (2009). p53 Isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish. Genes Dev 23: 278–290.

  7. Chrisanthar R, Knappskog S, Lokkevik E, Anker G, Ostenstad B, Lundgren S et al. (2008). CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer. PLoS One 3: e3062.

  8. Dohner K, Schlenk RF, Habdank M, Scholl C, Rucker FG, Corbacioglu A et al. (2005). Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 106: 3740–3746.

  9. Estey E, Dohner H . (2006). Acute myeloid leukaemia. Lancet 368: 1894–1907.

  10. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. (2005). Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 352: 254–266.

  11. Fenaux P, Preudhomme C, Quiquandon I, Jonveaux P, Lai JL, Vanrumbeke M et al. (1992). Mutations of the P53 gene in acute myeloid leukaemia. Br J Haematol 80: 178–183.

  12. Fridman JS, Lowe SW . (2003). Control of apoptosis by p53. Oncogene 22: 9030–9040.

  13. Fujita K, Mondal AM, Horikawa I, Nguyen GH, Kumamoto K, Sohn JJ et al. (2009). p53 isoforms delta133p53 and p53beta are endogenous regulators of replicative cellular senescence. Nat Cell Biol 11: 1135–1142.

  14. Gale RE, Hills R, Kottaridis PD, Srirangan S, Wheatley K, Burnett AK et al. (2005a). No evidence that FLT3 status should be considered as an indicator for transplantation in acute myeloid leukemia (AML): an analysis of 1135 patients excluding acute promyelocytic leukemia from the UK MRC AML10 and 12 trials. Blood 106: 3658–3665.

  15. Gale RE, Hills R, Pizzey AR, Kottaridis PD, Swirsky D, Gilkes AF et al. (2005b). Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia. Blood 106: 3768–3776.

  16. Gjertsen BT, Cressey LI, Ruchaud S, Houge G, Lanotte M, Doskeland SO . (1994). Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line. J Cell Sci 107: 3363–3377.

  17. Gjertsen BT, Oyan AM, Marzolf B, Hovland R, Gausdal G, Doskeland SO et al. (2002). Analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses. J Hematother Stem Cell Res 11: 469–481.

  18. Graupner V, Schulze-Osthoff K, Essmann F, Janicke RU . (2009). Functional characterization of p53beta and p53gamma, two isoforms of the tumor suppressor p53. Cell Cycle 8: 1238–1248.

  19. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. (1998). The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children′s Leukaemia Working Parties. Blood 92: 2322–2333.

  20. Grisendi S, Mecucci C, Falini B, Pandolfi PP . (2006). Nucleophosmin and cancer. Nat Rev Cancer 6: 493–505.

  21. Haferlach C, Dicker F, Herholz H, Schnittger S, Kern W, Haferlach T . (2008). Mutations of the TP53 gene in acute myeloid leukemia are strongly associated with a complex aberrant karyotype. Leukemia 22: 1539–1541.

  22. Hainaut P . (1995). The tumor suppressor protein p53: a receptor to genotoxic stress that controls cell growth and survival. Curr Opin Oncol 7: 76–82.

  23. Hall PA, McCluggage WG . (2006). Assessing p53 in clinical contexts: unlearned lessons and new perspectives. J Pathol 208: 1–6.

  24. Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT et al. (2004). Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118: 217–228.

  25. Irish JM, Anensen N, Hovland R, Skavland J, Borresen-Dale AL, Bruserud O et al. (2007). Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53. Blood 109: 2589–2596.

  26. Kojima K, Vickers SM, Adsay NV, Jhala NC, Kim HG, Schoeb TR et al. (2007). Inactivation of Smad4 accelerates Kras(G12D)-mediated pancreatic neoplasia. Cancer Res 67: 8121–8130.

  27. Komeno Y, Kurokawa M, Imai Y, Takeshita M, Matsumura T, Kubo K et al. (2005). Identification of Ki23819, a highly potent inhibitor of kinase activity of mutant FLT3 receptor tyrosine kinase. Leukemia 19: 930–935.

  28. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA et al. (2001). The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 98: 1752–1759.

  29. Levine AJ . (1997). p53, the cellular gatekeeper for growth and division. Cell 88: 323–331.

  30. Levine AJ, Oren M . (2009). The first 30 years of p53: growing ever more complex. Nat Rev Cancer 9: 749–758.

  31. Lonning PE . (2003). Study of suboptimum treatment response: lessons from breast cancer. Lancet Oncol 4: 177–185.

  32. Löwenberg B, Downing JR, Burnett A . (1999). Acute myeloid leukemia. N Engl J Med 341: 1051–1062.

  33. Löwenberg B, van Putten W, Theobald M, Gmur J, Verdonck L, Sonneveld P et al. (2003). Effect of priming with granulocyte colony-stimulating factor on the outcome of chemotherapy for acute myeloid leukemia. N Engl J Med 349: 743–752.

  34. Löwenberg B . (2008). Acute myeloid leukemia: the challenge of capturing disease variety. Hematology Am Soc Hematol Educ Program 1–11.

  35. Mayer RJ, Davis RB, Schiffer CA, Berg DT, Powell BL, Schulman P et al. (1994). Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B. N Engl J Med 331: 896–903.

  36. Pardee TS, Zuber J, Lowe SW . (2011). Flt3-ITD alters chemotherapy response in vitro and in vivo in a p53-dependent manner. Exp Hematol 39: 473–485 e474.

  37. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P et al. (2008). An integrated genomic analysis of human glioblastoma multiforme. Science 321: 1807–1812.

  38. Petitjean A, Achatz MI, Borresen-Dale AL, Hainaut P, Olivier M . (2007). TP53 mutations in human cancers: functional selection and impact on cancer prognosis and outcomes. Oncogene 26: 2157–2165.

  39. Preudhomme C, Fenaux P . (1997). The clinical significance of mutations of the P53 tumour suppressor gene in haematological malignancies. Br J Haematol 98: 502–511.

  40. Rohaly G, Chemnitz J, Dehde S, Nunez AM, Heukeshoven J, Deppert W et al. (2005). A novel human p53 isoform is an essential element of the ATR-intra-S phase checkpoint. Cell 122: 21–32.

  41. Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF et al. (2005). Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 106: 3733–3739.

  42. Schottelius A, Brennscheidt U, Ludwig WD, Mertelsmann RH, Herrmann F, Lubbert M . (1994). Mechanisms of p53 alteration in acute leukemias. Leukemia 8: 1673–1681.

  43. Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A et al. (2009). Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461: 809–813.

  44. Srinivasa SP, Doshi PD . (2002). Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways cooperate in mediating cytokine-induced proliferation of a leukemic cell line. Leukemia 16: 244–253.

  45. Tallman MS, Gilliland DG, Rowe JM . (2005). Drug therapy for acute myeloid leukemia. Blood 106: 1154–1163.

  46. Van Belle W, Anensen N, Haaland I, Bruserud O, Hogda KA, Gjertsen BT . (2006). Correlation analysis of two-dimensional gel electrophoretic protein patterns and biological variables. BMC Bioinform 7: 198.

  47. Verhaak RG, Goudswaard CS, van Putten W, Bijl MA, Sanders MA, Hugens W et al. (2005). Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood 106: 3747–3754.

  48. Vousden KH, Lane DP . (2007). p53 in health and disease. Nat Rev Mol Cell Biol 8: 275–283.

  49. Wahlin A, Brinch L, Hornsten P, Evensen SA, Oberg G, Simonsson B et al. (1997). Outcome of a multicenter treatment program including autologous or allogeneic bone marrow transplantation for de novo acute myeloid leukemia. Eur J Haematol 58: 233–240.

  50. Wheatley K, Burnett AK, Goldstone AH, Gray RG, Hann IM, Harrison CJ et al. (1999). A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council′s Adult and Childhood Leukaemia Working Parties. Br J Haematol 107: 69–79.

  51. Yeh PY, Chuang SE, Yeh KH, Song YC, Chang LL, Cheng AL . (2004). Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death. Oncogene 23: 3580–3588.

Download references


This study was supported by grants from the Research Council of Norway's National Program for Research in Functional Genomics, the Western Norway Regional Health Authority and the Norwegian Cancer Society.

Author information



Corresponding author

Correspondence to B T Gjertsen.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ånensen, N., Hjelle, S., Van Belle, W. et al. Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia. Oncogene 31, 1533–1545 (2012).

Download citation


  • p53 protein isoform
  • p53 β
  • p53 γ
  • acute myeloid leukemia/NPM1/FLT3-ITD

Further reading