Implications of Gene and p53 Protein Alterations in Determining Progression of Chronic Myeloid Leukemia Phases.

Abstract TP53 gene and p53 protein play an important role in the control of the cell cycle and DNA repair. Alterations in the TP53 gene and in the p53 protein expression have been considered an unfavorable prognostic factor for certain forms of human cancer. Mutations in the TP53 gene in patients with chronic myeloid leukemia (CML) were found in up to 30 %, specially among those in blastic crisis. At present, the only widely available technology that reliable detects and defines all mutations is DNA sequencing. However, the routine of sequencing the entire TP53 gene, in all suggestive cases of mutation, in the laboratorial routine is prohibitively costly, complex, and time consuming. To screening of those genetic alterations for p53 protein expression and TP53 gene abnormalities, in CML patients, flow cytometry (FC) and single strand conformation polymorphism of polymerase chain reaction products (PCR-SSCP) techniques are proposed. We report the results of an analysis of 72 blood samples from CML patients: 54 in chronic phase (33 in initial chronic phase (ICP) and 21 in late chronic phase (LCP)), 7 in accelerated phase (AP) and 11 in blastic crisis (BC). DNA structure for exons 5, 6, 7, 8 and 8–9 of the TP53 gene and p53 protein expression using the monoclonal antibody DO7 by FC was performed. By PCR-SSCP analysis, shift in eletrophoretic mobility of the TP53 gene, were detected in 11 patients and p53 protein were expressed in 17 out of 72 CML patients. The abnormal PCR-SSCP pattern were showed in one case of ICP (exon 7), five cases of LCP (two in the exon 7, one in the exon 8 and two in the exons 8–9), one in AP (exon 8) and four in BC (two in the exon 5, one in the exon 6 and one in the exon 8–9). In these cases, the p53 protein were expressed in seven cases (in all cases of CB and AC and 2 out of 5 LCP samples of CML patients). The statistic analysis by qui-square test showed significance between the results of the two methods (p= 0,002). These results suggest that the two concomitant methods can increase the sensibility for screening the p53 protein expression and TP53 gene abnormalities in CML patients. The presence of abnormal profile of PCR-SSCP associated to the p53 protein expression, in advanced phases of this disease, might be considered an important indicator of the phase progression of CML.

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Genetic alterations of the TP53 gene, p53 protein expression and hpv infection in primary cervical carcinomas

The Journal of Pathology ◽

10.1002/path.1711710207 ◽

1993 ◽

Vol 171 (2) ◽

pp. 105-114 ◽

Cited By ~ 50

Author(s):

Åslaug Helland ◽

Ruth Holm ◽

Gunnar Kristensen ◽

Janne Kaern ◽

Frank Karlsen ◽

...

Keyword(s):

Protein Expression ◽

P53 Protein ◽

Tp53 Gene ◽

Hpv Infection ◽

Genetic Alterations ◽

Cervical Carcinomas ◽

P53 Protein Expression

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JunB gene expression is inactivated by methylation in chronic myeloid leukemia

Blood ◽

10.1182/blood-2002-05-1598 ◽

2003 ◽

Vol 101 (8) ◽

pp. 3205-3211 ◽

Cited By ~ 53

Author(s):

Ming-Yu Yang ◽

Ta-Chih Liu ◽

Jan-Gowth Chang ◽

Pai-Mei Lin ◽

Sheng-Fung Lin

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mutational Analysis ◽

Blast Crisis ◽

Chronic Phase ◽

Polymerase Chain Reaction Analysis ◽

Myeloproliferative Disease ◽

Coding Region ◽

Blastic Crisis ◽

Cpg Sites

Abstract JunB is a component of the Jun family genes of the activating protein-1 transcription factors that are important in the control of cell growth and differentiation and neoplastic transformation. Recently, it was demonstrated that transgenic mice specifically lacking JunB expression in the myeloid lineage developed a myeloproliferative disease, eventually progressing to blast crisis that resembled human chronic myeloid leukemia (CML). To gain further insights into the role of JunB in human CML, we examined peripheral blood from 17 healthy individuals and CML patients (11 in blastic crisis and 21 in chronic phase) by real-time quantitative reverse transcription–polymerase chain reaction analysis for the expression of JunB. The results showed the expression levels of JunB were significantly impaired in CML cases (blastic crisis < chronic phase < normal). Mutational analysis of the whole gene and methylation analysis of cytosine-phosphate guanosine (CpG) sites at the promoter area were further performed to investigate the possible mechanisms. However, no mutation was found within the coding region or the 9 flanking evolutionarily conserved regions in all CML cases. Interestingly, in the promoter area of JunB gene, most of the CpG sites were methylated in CML cases; in contrast, none of these CpG sites were methylated in normal cases. Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of JunB expression. Our results suggest that the down-regulated JunB expression in CML was due to the inactivation of JunB gene by methylation and the differential expression was correlated to the ratio of cells being methylated.

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High P53 Protein Expression Level Independent of Mutational Status Is An Adverse Prognostic Factor for Survival in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.1490.1490 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1490-1490 ◽

Cited By ~ 1

Author(s):

Alfonso Quintás-Cardama ◽

Yi Hua Qiu ◽

Sean Post ◽

Steven M. Kornblau

Keyword(s):

Overall Survival ◽

Protein Expression ◽

Myeloid Leukemia ◽

P53 Protein ◽

P53 Mutations ◽

P53 Expression ◽

The Poor ◽

P53 Protein Expression ◽

Mutational Status ◽

Acute Myeloid

Abstract Abstract 1490 Background: The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML). In AML, p53 mutations have been associated with poor risk cytogenetics (i.e. complex karyotype, −5/−7). However, the function of p53 can also be compromised by protein stabilization and/or expression. The implications of p53 protein expression have not been studied in AML. Methodology: We assessed p53 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts (719 samples). Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1: 2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status was determined by Sanger sequencing of exons 5 through 9 of the p53 gene. Results: Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). Cases of AML-M3 and –M6 exhibited higher expression of p53 than other FAB subtypes. p53 expression directly correlated with age (p=0.01) and CD34 (p=0.001) and inversely correlated with WBC (p=0.007), BM (p=0.0001) and PB (p=0.0001) blasts, platelets (p=0.007), HLA-DR (p=0.01), CD19 (p=0.02), and survival (p=0.01). High p53 (p53high) expression level was more associated with unfavorable cytogenetics than with favorable or intermediate cytogenetics (p=0.00001). When all cytogenetic abnormalities were considered, pts with −5 had the highest levels of p53 (p=0.00001). Pts with RAS mutations, but not those with FLT3-ITD, NPM1, or IDH1/2, had lower levels of p53 protein. When pts were divided according to the level of p53 protein expression p53high was associated with lower complete remission (CR) rates (51% vs 56%; p=??) and higher relapsed rates (82% vs 62%; p=??). The median overall survival (OS) of pts with p53high and p53low were 29.8 vs. 51 wks (p=0.009). Most cases with p53high had unfavorable cytogenetics and the effect on OS was predominantly seen in that subpopulation with p53high and p53low pts living a medina of 23.4 vs. 36 wks (p=0.07), respectively. In order to determine whether the poor outcomes associated with p53high were due to the presence of a higher rate of p53 mutations among pts with p53high, we determined the p53 mutational status of 55 pts. p53high was highly correlated with the presence of p53 mutations as the latter were detected in 17/40 pts with p53high but in only 1/16 pts with p53low. Importantly, the presence of p53high, both in the presence (29 wks) or in the absence (24 wks) of p53 mutations, was associated with significantly worse overall survival compared with pts with p53low (56 wks; p=0.05, Figure 1). Multivariate analysis indicated that p53 is a significant independent risk factor for survival in AML. The final model included: age (p=0.000001), favorable cytogenetics (0.01), unfavorable cytogenetics (p=0.00001), WBC (p=0.0005), albumin (p=0.0003), FLT3-ITD (P=0.04), and P53 (P=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), c-MET (p=0.01), FoxO3a (p=0.004), KIT (p=0.001), p38p180p182 (p0.02), BAD (p=0.0001), cleaved PARP (p=0.002), cleaved PARP (p=0.01), TCF4 (p=0.02), fibronectin (p=0.02), and hsp70 (p=0.003), and negatively with AKTp473 (p=0.01), ERK (p=0.002), mTOR (p=0.005), PI3Kp85 (p=0.002), PKCδ (p=0.00002), GAB2 (p=0.00005), beclin (p=0.007), JMJD6 (p=0.001), Gata3 (p=0.02), p21 (p=0.01), and Mdm2 (p=0.001). Conclusions: Our results suggest that high levels of p53 protein constitute a powerful marker of short survival in AML. This effect is independent of p53 mutational status. The poor outcome of pts with high level of expression of p53 in the absence of p53 mutations suggests that the p53 pathway may be functionally perturbed in a much higher proportion of pts with AML than previously recognized. These data support the use of p53 protein expression levels in prognostication and in the development of targeted therapeutics. Disclosures: No relevant conflicts of interest to declare.

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Downregulation of Circadian Genes, PER1, PER2, and PER3, in Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v104.11.4317.4317 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4317-4317

Author(s):

Lin Sheng-Fung ◽

Ming-Yu Yang ◽

Jan-Gowth Chang ◽

Hui-Hua Hsiao ◽

Yi-Chang Liu ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Healthy Individuals ◽

Methylation Analysis ◽

Blastic Crisis ◽

Circadian Genes ◽

Expression Levels ◽

Cpg Sites ◽

Central Pacemaker

Abstract Circadian rhythm is present in all eukaryotic and some prokaryotic life forms. This time-keeping system is organized in a hierarchical fashion and composed of a self-sustained central pacemaker in the suprachiasmatic nuclei of the anterior hypothalamus and peripheral oscillators in most body cells. A recent study demonstrated that mice deficient in the clock gene mPer2 are cancer prone and display a deregulated temporal expression genes involved in cell cycle regulation, such as c-Myc, Cyclin D1, Cyclin A, and Mdm-2. These mice display salivary gland hyperplasia, and a large portion of them develops lymphomas. The results revealed that Per2 is an essential circadian gene and it is associated with proliferation control in mammals. In a more recent study, it was demonstrated that an age-dependent decay of the circadian clock both at the behavioral and the molecular levels was observed in mPer1−/−mCry2−/− double-mutant mice. To gain further insights into the roles of circadian genes in chronic myeloid leukemia (CML), we analyzed peripheral blood from 21 healthy individuals and 35 CML patients (18 in blastic crisis and 17 in chronic phase) by real-time quantitative RT-PCR for the expression of circadian genes PER1, PER2, and PER3. In blastic crisis cases, the expression levels of all three PER genes were significantly impaired than in healthy individuals (PER1, 1:5.97, p < 0.005; PER2, 1:13.51, p < 0.0001; PER3, 1:2181.33; p < 0.0001); whereas, in chronic phase, only the expression levels of PER2 and PER3 were significantly impaired (PER1, 1:1.23, p > 0.5; PER2, 1:2.47, p < 0.05; PER3, 1:14.22; p < 0.0001). Mutational analysis of the whole gene and methylation analysis of CpGs sites at the promoter area were further performed to investigate the possible mechanisms. No mutation was found within the coding regions of the three PER genes in all CML cases. Methylation analysis using methylation-specific PCR and direct sequencing showed no methylation in the promoter areas of both PER1 and PER2 genes. In contrast, most of the CpG sites were methylated in the promoter area of PER3 gene in CML cases and none of these CpG sites were methylated in healthy individual cases. In addition, the methylated CpG frequencies of PER3 gene differed in patients at blastic crisis and at chronic phase (CpG, 8.24 ± 0.73 vs. 4.48 ± 0.48, p < 0.0001; T/CpG, 10.47 ± 0.67 vs. 14.67 ± 0.46, p < 0.0001; TG, 1.24 ± 0.32 vs. 0.81 ± 0.16, p > 0.05). Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of PER3 expression. Our results suggest that the downregulated PER3 expression in CML was due to the inactivation of PER3 gene by methylation and the differential expression was correlated to the ratio of methylated CpG sites at the promoter region. We hope to explore the role of circadian genes in the tumorigenesis of leukemia and to establish circadian genes as novel and useful markers for diagnosis and references for therapies in leukemia.

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