Expression of a non‐functional p53 affects the sensitivity of cancer cells to gemcitabine

Abstract

Gemcitabine is a relatively new agent with promising activity in solid tumors. Few data are available regarding mechanisms of resistance to gemcitabine downstream from the drug-target interaction. The present study was performed to gain insight into the role of p53 status on the cytotoxicity of gemcitabine on cancer cells. Drug sensitivity, drug metabolism, cell kinetics and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the wildtype p53 (wt-p53) containing MN-1 cell line and, the MDD2 line containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to gemcitabine cytotoxicity than the MN-1 cell line. The resistant phenotype could not be attributed to a defective gemcitabine activation/degradation pathway or altered levels of expression of intracellular targets. Although both cell lines exhibited p53 accumulation, MN-1 but not MDD2 cells, displayed p21 WAF1 induction after exposure to gemcitabine. Gemcitabine induced an S-phase arrest in both cell lines. A more pronounced block in G1 phase, however, was observed in MN1 cells. Exposure to gemcitabine induced a higher degree of apoptosis in MN-1 than in MDD2 cells. This corresponded with suppression of Bcl-2 and Bcl-X/L expression in wt-p53 cells exposed to gemcitabine whereas Bcl-2 levels remained stable and Bcl-X/L levels increased in mut-p53 cells exposed to gemcitabine. We conclude that the p53 status of cancer cells influences their sensitivity to gemcitabine cytotoxicity. Our evidence suggests that loss of p53 function leads to loss of cell cycle control and alterations in the apoptotic cascade, conferring resistance to gemcitabine in cancer cell lines displaying a mut-p53. © 2002 Wiley-Liss, Inc. Key words: protein p53; drug resistance; gemcitabine; antimetabolites; cell death; apoptosisGemcitabine (difluorodeoxycytidine; dFdC) is an agent with promising activity in solid tumors. [1][2][3] This compound is a deoxycytidine analog with two fluorine substitutes for the two hydrogen atoms in the 2Ј position of the deoxyribose sugar. Gemcitabine activity is dependent upon the formation of a triphosphorylated metabolite that is subsequently incorporated into DNA.Gemcitabine enters the cell via a nucleoside transport system 4,5 and is phosphorylated into dFd-CMP by deoxycytidine kinase (dCK). 6 It is then subsequently phosphorylated by monophosphoand diphospho-pyrimidine kinases to the active 5Ј-diphosphate (dFd-CDP) and triphosphate (dFd-CTP) derivatives. 7 Inactivation of gemcitabine can occur by deamination or dephosphorylation. The conversion of gemcitabine by deamination into its inactive metabolite dFd-U is catalyzed by cytidine deaminase (CDD). 8 Cytoplasmic 5Ј nucleotidase (5NT) activity opposes that of dCK by dephosphorylating dFd-CMP, thereby preventing the production of the active form. 9 dFd-CTP is incorporated into DNA by replication synthesis in the C sites of the growing DNA strand. 10 Once incorporated into the DNA strand, an additional natu...

Role of NF-κB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death

“…PancTu-1 cells showed only a moderate increase in apoptotic cell death under these conditions, whereas BxPc-3 and Capan-1 cells exhibited almost no induction of apoptosis. Cell cylce analysis revealed a gain of cells in G1/early S phase in all cell lines (data not shown), consistent with one of the actions of gemcitabine as an inhibtor of DNA elongation (Ng et al, 2000;Galmarini et al, 2002). This indicated that the uptake or intracellular targeting of gemcitabine was not altered in these cell lines, thus excluding a failure of drug uptake or of metabolism as being responsible for the resistance of these cell lines against gemcitabine.…”

The cytotoxicity of chemotherapy drugs varies in cervical cancer cells depending on the p53 status

“…24,25 Similarly, MCF-7 cells and several pancreatic cancer cell lines expressing wt p53 were more susceptible to gemcitabine than the respective cell lines lacking active p53. 26,27 Although there are exceptions, each of the above studies is an example of a clear trend. However the role of p53 in etoposide-induced cytotoxicity is less clear, as some studies suggest a proapoptotic role for p53, especially in lymphoma and leukemia cells, 28,29 whereas other studies have concluded the opposite, for example with H1299 lung cancer cells.…”

Intrinsic Chemoresistance to Gemcitabine Is Associated with Decreased Expression of BNIP3 in Pancreatic Cancer

“…Another group comprises the genes involved in cell cycle regulation, proliferation, or apoptosis. Mutated p53 (13) and Bcl-xl (14,15) have been identified as possible molecular markers for gemcitabine chemoresistance, and both are directly involved in apoptosis. In addition, c-Src (16,17) and focal adhesion kinase (FAK; ref.…”