Konference: 2013 18th Congress of the European Hematology Association - účast ČR

Kategorie: Maligní lymfomy a leukémie

Téma: Chronic lymphocytic leukemia - Translational Research

Číslo abstraktu: P084

Autoři: Mgr. Veronika Navrkalová, Ph.D.; RNDr. Ludmila Šebejová; Mgr. Bc. Blanka Kubešová; prof. MUDr. Michael Doubek, Ph.D.; prof. MUDr. Jiří Mayer, CSc.; prof. RNDr. Šárka Pospíšilová, Ph.D.; Doc. MUDr. Martin Trbušek, PhD


Abnormalities of ATM gene are frequent in chronic lymphocytic leukemia (CLL) patients and represent important prognostic factor. ATM defects are commonly assessed through 11q deletion (11q-) monitoring, nevertheless complete ATM inactivation stems from biallelic ATM defects (mutation/11q- or two mutations) or from sole ATM mutation manifesting dominant-negative effect. After DNA damage caused by ionizing radiation (IR), it is well established that ATM plays crucial role in response to dsDNA breaks (DSBs), especially through triggering the p53 pathway. The impact of ATM inactivation in response to conventionally used DNA damaging drugs is much less understood although this inactivation could have important predictive value.


Our aim was to assess ATM inactivation impact on in vitro CLL cells response to fludarabine and doxorubicin, with respect to the ATM-p53 pathway activation.


To assess p53 pathway activation after drug exposure we used: (a) Real-time PCR to analyze p53-downstream gene (CDKN1A (p21), BBC3 (PUMA), BAX, and GADD45) induction after 24 h treatment of CLL cells with fludarabine and doxorubicin or after2, 10, 24 h IR exposure (5 Gy in total, 0.3 Gy/min), (b) Western blotting (WB) for total p53 and Ser15-p53 after 24 h fludarabine treatment; these experiments were performed on wt samples with artificially inactivated ATM (inhibitor KU55933) and on CLL samples harboring ATMmutation( s), and (c) WB for Ser1981-ATM and Ser15-p53 to evaluate immediate ATM activation in mutated samples after 1 h IR exposure.


The samples with inactivated ATM exhibited clearly impaired induction of all four p53-downstream genes after doxorubicin, but not fludarabine treatment (3/3 artificially inactivated samples and 16/20 ATM-mutated samples from our previous study; 14 samples harbored biallelic defect and two harbored single mutation at hot-spot codon 3008). Since doxorubicin is supposedly radiomimetic drug, we verified the ATM-dependent response to DSBs using IR. We performed the same analysis on 8 ATM-mutated samples and 5 samples with preserved ATM function (i.e., wt or sole 11q-) and observed similarly impaired response in ATM-defective group, however with the exception of BAX gene that had preserved induction. The preserved p53-downstream pathway response after fludarabine prompted us to analyze p53 accumulation and activation. Firstly, we confirmed that fludarabine creates DSBs (gH2AX accumulation after 24 h exposure) and then showed that in relevant proportion of cases the p53 stabilization is present (2/4 artificially inactivated samples and 3/4 ATM-mutated samples; the last mutated sample demonstrated partial p53 stabilization). Our observations show subtle ATM impact on studied response to fludarabine suggesting the involvement of other signaling kinase(s) in the p53 pathway activation. To further analyze loss of ATM function within the DNA damage response cascade, we also monitored ATM autophosphorylation on Ser1981 and p53 phosphorylation on Ser-15 in two ATM-mutated samples after IR. One ATM-mutant showed obviously diminished autophosphorylation and both mutants completely lost any activity towards p53 activation and stabilization.

Summary / Conclusion:

CLL cells lacking ATM activity manifest clearly impaired p53 pathway activation after doxorubicin, while this response appears to be normal after fludarabine. It seems that ATM inactivation is prominently manifested in the end of ATM-p53 pathway while initial processes are less influenced.

The work was supported by grants FR-TI2/254, NT13519 and MUNI/A/0723/2012.

Abstrakta v časopise Haematologica 2013, Suppl1

Online Program

Datum přednesení příspěvku: 14. 6. 2013