A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells

Abstract

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. ' 2006 Wiley-Liss, Inc.Key words: paclitaxel; cyclin-dependent kinase inhibitor p21; antineoplastic agent resistance Paclitaxel is one of the most effective anticancer drugs used in the clinic to treat a variety of solid tumors. In breast cancer patients, administration of paclitaxel in combination or sequentially with other chemotherapy drugs for the treatment of metastatic disease demonstrated to have more anticancer activity than previous nonpaclitaxel chemotherapy regimens. 1 Moreover, recent clinical trials also demonstrated that the use of paclitaxel as adjuvant therapy in early stage breast cancer patients helps to prevent relapse after the initial primary surgical treatment. 2,3 Despite paclitaxel increasing use, especially in therapy of curable cancers, no data are currently available on its effects on noncancerous human breast epithelial cells. This prompted us to expose hTERT-immortalized human mammary epithelial cells to a stringent selection by this agent in a 7-day drug exposure. We then analyzed the emergence of surviving cells in parallel populations. In contrast to prevalent cellular models of drug resistance derived by multiple stepwise selections, the PSP in our model have experienced a single paclitaxel treatment at concentration levels and time exposure similar than those used in clinical practice. In these experiments, mdr-1 expression was found to be positive by RT-PCR in 2 of 20 PSP (10%). Given the fact that the mdr-1-negative PSP displayed resistance to paclitaxel, we sought to determine whether other alterations existed in these PSP. Our findings indicate that mutations of the p21/WAF1 gene are involved in resistance to this agent. Material and methods ReagentsPaclitaxel was obtained from Bristol-Myers Squibb (Paris, France). Antibodies against C-terminal-p21/WAF1 (SX-118), p53 (DO7...

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma

“…Selective screening of patient DNA was performed as determined by its corresponding homozygosity profile. Mutation analysis was carried out by PCR-direct sequence analysis of selected regions of protein tyrosine phosphatase, non-receptor type 11 (PTPN11) (exons 3 and 13); 26 histone H4 (HIST1H4D), Splicing factor, arginine/serine-rich 3 (SFRS3), cyclin-dependent kinase inhibitor 1A (CDKN1A/p21 WAF1/CIP1 ), 27 cyclin D3 (CCND3) and cyclin-dependent kinase inhibitor 2A (CDKN2A) were all screened in their entirety. Tumour protein 53 (TP53) screening of this series has been published previously.…”

Paclitaxel resistance in untransformed human mammary epithelial cells is associated with an aneuploidy-prone phenotype

“…Paclitaxelsurviving populations were obtained from HME1 cells as reported previously (Galmarini et al, 2006). Briefly, HME1 cells were seeded onto 20 separate 96-well plates at a concentration of 100 cells/well.…”

“…These assays were carried out without the use of specific markers of chromosomes, and thus estimated the global missegregation process but did not specify associated chromosome aberrations (for example, nucleoplasmic bridges, chromosome non-disjunction and breakage). We decided to test effects of 1 and 20 nM paclitaxel on chromosome segregation, as these two doses were previously found to be the paclitaxel IC 20 and IC 80 in HME1 cell line (Galmarini et al, 2006). Moreover, these doses act differentially to induce cell death in untransformed mammary cells.…”

“…However, despite its increasing use, almost no data are currently available concerning its effects in normal human mammary epithelial cells. In a precedent work, we described that sub-cell lines of untransformed human mammary epithelial cells (HME1) were able to survive to a 1-week paclitaxel treatment (paclitaxel-surviving populations, PSPs) (Galmarini et al, 2006). In most of these sublines, the emergence of a transitory or stable drug resistance phenotype was related to the inactivation of p21/WAF1 protein.…”

P21/WAF1 mutation and drug resistance to paclitaxel in locally advanced breast cancer

“…1 The authors reported a point mutation in exon 2 of the p21 gene (WAF1, CIP1, CDKN1A), leading to a premature STOP in codon 127 and paclitaxel resistance in noncancerous epithelial breast cells. The cyclin-dependent kinase inhibitor p21 is a main effector of growth arrest induced by p53.…”