2006
Rituximab Senzitizes B-CLL Cells with Inactived p53 to fludarabine
TRBUŠEK, Martin, Soňa ČEJKOVÁ, Jitka CHUMCHALOVÁ, Zdeněk POSPÍŠIL, Jana ŠMARDOVÁ et. al.Základní údaje
Originální název
Rituximab Senzitizes B-CLL Cells with Inactived p53 to fludarabine
Název česky
Rituximab senzitivizuje B-CLL buňky s inaktivovaným p53 k fludarabinu
Autoři
TRBUŠEK, Martin (203 Česká republika, garant), Soňa ČEJKOVÁ (203 Česká republika), Jitka CHUMCHALOVÁ (203 Česká republika), Zdeněk POSPÍŠIL (203 Česká republika), Jana ŠMARDOVÁ (203 Česká republika), Petr KUGLÍK (203 Česká republika), Michael DOUBEK (203 Česká republika), Yvona BRYCHTOVÁ (203 Česká republika), Šárka POSPÍŠILOVÁ (203 Česká republika) a Jiří MAYER (203 Česká republika)
Vydání
2006
Další údaje
Jazyk
angličtina
Typ výsledku
Konferenční abstrakt
Obor
30200 3.2 Clinical medicine
Stát vydavatele
Spojené státy
Utajení
není předmětem státního či obchodního tajemství
Kód RIV
RIV/00216224:14310/06:00019160
Organizační jednotka
Přírodovědecká fakulta
UT WoS
000242440402092
Klíčová slova anglicky
B-CLL p53 rituximab fludarabine in vitro
Příznaky
Recenzováno
Změněno: 1. 4. 2010 18:31, prof. RNDr. Zdeněk Pospíšil, Dr.
V originále
Background: Defects in the p53 gene predispose B CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B CLL cells bearing p53 abnormalities. One reason for that might be a methodological: rituximab alone does not have virtually any effect on the viability of B CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53. Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B CLL cells. Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia 2006, 20: 1159 to 1161[Medline]). Vitally frozen samples were used in all cases, after 24h precultivation. Mononuclear cells were cultivated for 72h with or without 20ug/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40ug/ effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase3 cleavage, which was proved previously to occur in drug treated B CLL cells. Results: In the subgroup of eleven p53/wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp:Glu), one sample manifested with antagonistic effect (del / 220 Tyr:Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg:Gln, del / 249 Arg:Gly, del / del aa 252, del / del aa 252 to 254, and a composed mutant 281Asp:Asn / 254 Ile:Thr). We noticed a statistically significant potentiation also in three out of four ATM deleted samples (ATM is the p53regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase3 cleavage in some (but not all) samples. Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind. ml to 0,625ug/ml). The cell viability was determined by a WST1 assay. A sensitization
Česky
Background: Defects in the p53 gene predispose B CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B CLL cells bearing p53 abnormalities. One reason for that might be a methodological: rituximab alone does not have virtually any effect on the viability of B CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53. Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B CLL cells. Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia 2006, 20: 1159 to 1161[Medline]). Vitally frozen samples were used in all cases, after 24h precultivation. Mononuclear cells were cultivated for 72h with or without 20ug/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40ug/ effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase3 cleavage, which was proved previously to occur in drug treated B CLL cells. Results: In the subgroup of eleven p53/wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp:Glu), one sample manifested with antagonistic effect (del / 220 Tyr:Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg:Gln, del / 249 Arg:Gly, del / del aa 252, del / del aa 252 to 254, and a composed mutant 281Asp:Asn / 254 Ile:Thr). We noticed a statistically significant potentiation also in three out of four ATM deleted samples (ATM is the p53regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase3 cleavage in some (but not all) samples. Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind. ml to 0,625ug/ml). The cell viability was determined by a WST1 assay. A sensitization
Návaznosti
| LC06024, projekt VaV |
| ||
| NR8445, projekt VaV |
|