Bcl-2 protein expression and p53 gene mutation in chronic lymphocytic leukemia: correlation with in vitro sensitivity to chlorambucil and purine analogs.
OBJECTIVE
Bcl-2 oncogenic protein expression plays a major role in blocking the apoptotic mechanism. p53 gene mutations have also been suggested to account for the chlorambucil resistance in CLL. Thus we studied the relationship between bcl-2 protein expression, p53 gene mutations and in vitro drug sensitivity in CLL.


METHODS
Fifty-three samples from untreated CLL patients in early disease stages were tested in vitro for chemosensitivity to chlorambucil (CLB), fludarabine (FAMP) and 2-chlorodeoxyadenosine (2-CDA) using the MTT assay. Intracellular bcl-2 protein expression was evaluated by flow cytometry analysis. p53 gene mutations were detected by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis.


RESULTS
The median LD50 values were 1.55 microM, 4.41 microM and 58.2 microM for 2-CDA, FAMP and CLB, respectively. About 23%, 41% and 11% of samples were defined as being sensitive to FAMP, 2-CDA and CLB, respectively, when samples were clustered for LD50 threshold values corresponding to the plasmatic levels of the drug. No statistically significant difference in bcl-2 protein expression was noted between sensitive and resistant samples for each drug. A p53 gene mutation was detected in 4 of the 30 cases studies and all of them were among samples resistant to CLB.


CONCLUSIONS
Bcl-2 expression is not an indicator of in vitro response to drugs in CLL; similarly, although the four cases showing a p53 gene mutation were associated with CLB resistance, drug resistant samples were also observed in the group of patients showing wild type p53, suggesting multiple mechanisms of drug resistance in CLL.
Morabito
F
F
Dipartimento di Emato-Oncologia, Azienda Ospedaliera Melacrino-Bianchi-Morelli, Reggio Calabria, Italy. sp0040@relay.it.net
Filangeri
M
M
Callea
I
I
Sculli
G
G
Callea
V
V
Fracchiolla
N S
NS
Neri
A
A
Brugiatelli
M
M
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D002699:Chlorambucil / Q000494:pharmacology*; D017338:Cladribine / Q000494:pharmacology*; D004273:DNA, Neoplasm / Q000032:analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D016158:Genes, p53; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000473:pathology*; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D018906:Antineoplastic Agents, Alkylating; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D002699:Chlorambucil; D017338:Cladribine; D014740:Vidarabine; C024352:fludarabine