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High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases.

D'Angelo V, Crisci S, Casale F, Addeo R, Giuliano M, Pota E, Finsinger P, Baldi A, Rondelli R, Abbruzzese A, Caraglia M, Indolfi P - J. Exp. Clin. Cancer Res. (2009)

Bottom Line: On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04).Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells.These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pediatric Oncology Service, Pediatric Department, F Fede, II University of Naples, Naples, Italy. velia.dangelo@unina2.it

ABSTRACT
Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC) and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (ALL) and stage IV non-Hodgkin Lymphoma (NHL). We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04). On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04). Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

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Western blot assay for the expression of pErk 1 and 2 and total Erk 1 and 2. The cells were processed for the determination of the phosphorylation and expression of Erk-1 and 2 evaluated after blotting with a specific anti-pMAPK and an anti-MAPK Mab, respectively, as described in "Materials and Methods". Expression of the house-keeping protein α-tubulin was used as loading control. In the same figure, the scores of the staining intensities of pErk-1 obtained at ICC in the same samples are also shown. The experiments were performed at least three different times and the results were always similar.
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Figure 2: Western blot assay for the expression of pErk 1 and 2 and total Erk 1 and 2. The cells were processed for the determination of the phosphorylation and expression of Erk-1 and 2 evaluated after blotting with a specific anti-pMAPK and an anti-MAPK Mab, respectively, as described in "Materials and Methods". Expression of the house-keeping protein α-tubulin was used as loading control. In the same figure, the scores of the staining intensities of pErk-1 obtained at ICC in the same samples are also shown. The experiments were performed at least three different times and the results were always similar.

Mentions: The statistical analysis of these data showed that the staining intensity of the four analyzed proteins directly correlated with the number of positive cells (p < 0.05). Moreover, we found a simultaneous activation of pJNK and Erk-1 in the evaluated blasts (r = 0.26; p = 0.025). In order to validate the results obtained in ICC, we have evaluated the expression of p-Erk-1 with western blotting with conventional antibodies used for the determination of p-Erk-1 and 2 and total Erk-1/2. The lower band shown in the gel, corresponding to a M.W. of 44 KDa, is clearly assessable in all the samples. The activity of the enzyme in the different evaluable patients strongly correlated to that one derived from experiments performed on blasts with ICC. An example of Erk-1 expression and activity on 10 different samples is now shown in Figure 2. Similar results were also obtained on all the other samples (data not shown).


High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases.

D'Angelo V, Crisci S, Casale F, Addeo R, Giuliano M, Pota E, Finsinger P, Baldi A, Rondelli R, Abbruzzese A, Caraglia M, Indolfi P - J. Exp. Clin. Cancer Res. (2009)

Western blot assay for the expression of pErk 1 and 2 and total Erk 1 and 2. The cells were processed for the determination of the phosphorylation and expression of Erk-1 and 2 evaluated after blotting with a specific anti-pMAPK and an anti-MAPK Mab, respectively, as described in "Materials and Methods". Expression of the house-keeping protein α-tubulin was used as loading control. In the same figure, the scores of the staining intensities of pErk-1 obtained at ICC in the same samples are also shown. The experiments were performed at least three different times and the results were always similar.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664791&req=5

Figure 2: Western blot assay for the expression of pErk 1 and 2 and total Erk 1 and 2. The cells were processed for the determination of the phosphorylation and expression of Erk-1 and 2 evaluated after blotting with a specific anti-pMAPK and an anti-MAPK Mab, respectively, as described in "Materials and Methods". Expression of the house-keeping protein α-tubulin was used as loading control. In the same figure, the scores of the staining intensities of pErk-1 obtained at ICC in the same samples are also shown. The experiments were performed at least three different times and the results were always similar.
Mentions: The statistical analysis of these data showed that the staining intensity of the four analyzed proteins directly correlated with the number of positive cells (p < 0.05). Moreover, we found a simultaneous activation of pJNK and Erk-1 in the evaluated blasts (r = 0.26; p = 0.025). In order to validate the results obtained in ICC, we have evaluated the expression of p-Erk-1 with western blotting with conventional antibodies used for the determination of p-Erk-1 and 2 and total Erk-1/2. The lower band shown in the gel, corresponding to a M.W. of 44 KDa, is clearly assessable in all the samples. The activity of the enzyme in the different evaluable patients strongly correlated to that one derived from experiments performed on blasts with ICC. An example of Erk-1 expression and activity on 10 different samples is now shown in Figure 2. Similar results were also obtained on all the other samples (data not shown).

Bottom Line: On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04).Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells.These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pediatric Oncology Service, Pediatric Department, F Fede, II University of Naples, Naples, Italy. velia.dangelo@unina2.it

ABSTRACT
Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC) and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (ALL) and stage IV non-Hodgkin Lymphoma (NHL). We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04). On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04). Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

Show MeSH
Related in: MedlinePlus