A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.
We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.
Galmarini
Carlos María
CM
Unité d'Oncologie Moléculaire, Centre Léon Bérard, Lyon, France. fgalma@rockfeller.univ-lyon1.fr
Bouchet
Benjamin Pierre
BP
Audoynaud
Carole
C
Lamblot
Christelle
C
Falette
Nicole
N
Bertholon
Jacques
J
Wang
Qing
Q
Beghin
Anne
A
Dumontet
Charles
C
Puisieux
Alain
A
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D020137:Base Pair Mismatch; D015153:Blotting, Western; D003062:Codon; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000528:radiation effects; D004351:Drug Resistance / Q000187:drug effects / Q000528:radiation effects; D004847:Epithelial Cells / Q000187:drug effects; D005260:Female; D005786:Gene Expression Regulation; D020868:Gene Silencing; D019450:Genes, MDR / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000187:drug effects* / Q000528:radiation effects; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011829:Radiation Dosage; D011839:Radiation, Ionizing; D013912:Threonine
D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; D003062:Codon; D050759:Cyclin-Dependent Kinase Inhibitor p21; D013912:Threonine; D017239:Paclitaxel