Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

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...

The p53-transcriptional target, BTG2 TIS21/PC3 , was previously identified as an antiproliferative gene. However, the precise biological functions of the protein product remain to be elucidated. BTG2 TIS21/PC3 expression is induced in vivo during neurogenesis, and the gene is transiently expressed in vitro in rat pheochromocytoma PC12 cells after induction of neuronal differentiation by addition of nerve growth factor (NGF). These observations suggest that BTG2 TIS21/PC3 is functionally significant during the neuronal differentiation process. To test this hypothesis, a vector that expressed BTG2 TIS21/PC3 under the control of an inducible promoter was introduced into PC12 cells. Growth arrest and differentiation in response to NGF were greatly enhanced by BTG2 TIS21/PC3 overexpression. Furthermore, an antisense oligonucleotide complementary to BTG2 TIS21/PC3 mRNA, which was able to inhibit endogenous BTG2 TIS21/PC3 expression, triggered programmed cell death in differentiated PC12 cells. These observations confirm that BTG2 TIS21/PC3 expression promotes neuronal differentiation and that it is required for survival of terminally differentiated cells.

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