The p53 tumor suppressor protein inhibits the formation of tumors through induction of cell cycle arrest and/or apoptosis. In the present study we demonstrated that p53 is also a powerful inhibitor of human telomerase reverse transcriptase (hTERT), a key component for telomerase. Activation of either exogenous temperature-sensitive (ts) p53 in BL41 Burkitt lymphoma cells or endogenous wild type (wt) p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of hTERT mRNA expression, independently of the induction of the p53 target gene p21. Co-transfection of an hTERT promoter construct with wt p53 but not mutant p53 in HeLa cells inhibited the hTERT promoter activity. Furthermore, the activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. Finally, wt p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Since activation of telomerase, widely observed in human tumor cell lines and primary tumors, is a critical step in tumorigenesis, wt p53-triggered inhibition of hTERT/telomerase expression may re¯ect yet another mechanism of p53-mediated tumor suppression. Our ®ndings provide new insights into both the biological function of p53 and the regulation of hTERT/telomerase expression. Oncogene (2000) 19, 5123 ± 5133.

Background: Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. Aim: To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. Patients: 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. Methods: Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6-mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. Results: 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)); 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio; p = 0.02). Patients with meTIMP concentrations .11 450 pmol/8610 8 red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0; p = 0.015). Conclusions: After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity.

The purpose of this study was to evaluate the role of sequence variants in the CYP2C8, ABCB1 and CYP3A4 genes and the CYP3A4 phenotype for the pharmacokinetics and toxicity of paclitaxel in ovarian cancer patients. Thirty-eight patients were treated with paclitaxel and carboplatin. The genotypes of CYP2C8*1B, *1C, *2, *3, *4, *5, *6, *7, *8 and P404A, ABCB1 G2677T/A and C3435T, as well as CYP3A4*1B, were determined by pyrosequencing. Phenotyping of CYP3A4 was performed in vivo with quinine as a probe. The patients were monitored for toxicity and 23 patients underwent a more extensive neurotoxicity evaluation. Patients heterozygous for G/A in position 2677 in ABCB1 had a significantly higher clearance of paclitaxel than most other ABCB1 variants. A lower clearance of paclitaxel was found for patients heterozygous for CYP2C8*3 when stratified according to the ABCB1 G2677T/A genotype. In addition, the CYP3A4 enzyme activity in vivo affected which metabolic pathway was dominant in each patient, but not the total clearance of paclitaxel. The exposure to paclitaxel correlated to the degree of neurotoxicity. Our findings suggest that interindividual variability in paclitaxel pharmacokinetics might be predicted by ABCB1 and CYP2C8 genotypes and provide useful information for individualized chemotherapy.Paclitaxel in combination with carboplatin is the standard chemotherapy for ovarian cancer. Carboplatin doses are adjusted according to the renal function, whereas paclitaxel is used in standardized doses according to body surface area. The pharmacokinetics and the response to paclitaxel treatment vary greatly among individuals and one factor of importance for these differences might be the genetic variability. Our belief is that it would be important to be able to predict the highest yet safe starting dose for each individual to avoid undertreatment. Understanding the mechanisms behind the interindividual differences in the pharmacokinetics of paclitaxel should be the foundation for establishing individual dosages.It has been suggested that the pharmacokinetics of paclitaxel are affected by several proteins, such as metabolic enzymes and drug transporters [1]. Systemic elimination of paclitaxel occurs by hepatic metabolism involving the cytochrome P450 (CYP) enzymes, CYP3A4 and CYP2C8 [2]. Paclitaxel is converted to p -3 ′ -hydroxypaclitaxel by CYP3A4 and CYP2C8 catalyzes the formation of 6 α -hydroxypaclitaxel [3,4]. These metabolites can be further oxidized to 6 α -, p -3 ′ -dihydroxypaclitaxel [4,5]. All three metabolites are less potent than the parent compound in inhibiting cell growth in vitro [6,7]. Several single nucleotide polymorphisms (SNP) have been reported in the CYP2C8 gene and some alleles (*2, *3, *7, *8 and P404A) have been associated with decreased 6 α -hydroxypaclitaxel production in vitro [8][9][10][11]. The CYP2C8*5 allele, a premature stop-codon, is also expected to encode an inactive protein [12]. However, the effects of the polymorphisms on paclitaxel pharmacokinetics in vivo are still ...

Purpose: P-glycoprotein, encoded by the mdr-1 gene, confers multidrug resistance to a variety of antineoplastic agents, e.g., paclitaxel. Recently, different polymorphisms in the mdr-1 gene have been identified and their consequences for the function of P-glycoprotein, as well as for the treatment response to P-glycoprotein substrates, are being clarified. We analyzed the allelic frequencies at polymorphic sites G2677T/A and C3435T in ovarian cancer patients with good or poor response to treatment with paclitaxel in combination with carboplatin in order to evaluate their predictive values. Experimental Design: Fifty-three patients were included in the study; 28 of them had been relapse-free for at least 1 year and 25 had progressive disease or relapsed within 12 months. A reference material consisting of 200 individuals was also analyzed. The genotypes of each single nucleotide polymorphism (SNP) were determined using Pyrosequencing. Results:The G2677T/A SNP was found to significantly correlate with treatment outcome. The probability of responding to paclitaxel treatment was higher in homozygously mutated patients (T/T or T/A; Fisher's exact test; P < 0.05). The frequency of the T or A alleles was also higher in the group of patients who had a good response (P < 0.05). There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment (m 2 test for linear-by-linear association; P = 0.03). However, the C3435T SNP was not found to correlate to treatment outcome. Conclusions: The mdr-1 polymorphism G2677T/A in exon 21 correlates with the paclitaxel response in ovarian cancer and may be important for the function of P-glycoprotein and resistance to paclitaxel and provide useful information for individualized therapy.

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The primary purpose of this study was to evaluate the effect of CYP2C8*3 and three genetic ABCB1 variants on the elimination of paclitaxel. We studied 93 Caucasian women with ovarian cancer treated with paclitaxel and carboplatin. Using sparse sampling and nonlinear mixed effects modeling, the individual clearance of unbound paclitaxel was estimated from total plasma paclitaxel and Cremophor EL. The geometric mean of clearance was 385 l h -1 (range 176-726 l h -1 ). Carriers of CYP2C8*3 had 11% lower clearance than non-carriers, P ¼ 0.03. This has not been shown before in similar studies; the explanation is probably the advantage of using both unbound paclitaxel clearance and a population of patients of same gender. No significant association was found for the ABCB1 variants C1236T, G2677T/A and C3435T. Secondarily, other candidate single-nucleotide polymorphisms were explored with possible associations found for CYP2C8*4 (P ¼ 0.04) and ABCC1 g.7356253C4G (P ¼ 0.04).

Summary.In 95 leukaemic cell samples from 66 patients with acute myelogenous leukaemia (AML) (47 de novo and 19 secondary AML) telomerase activity was determined and the expression of the telomerase components: telomerase reverse transcriptase (hTERT), telomerase RNA template (hTR) and telomerase-associated protein (TP1) evaluated by RT-PCR. Compared to peripheral blood mononuclear cells (PBMC) from normal adult 87% (82/95) of patient samples exhibited elevated telomerase activity. hTERT, but not hTR and TP1 expression strongly correlated with the levels of telomerase activity (r ¼ 0·47, P < 0·0001). The levels of telomerase activity were significantly higher at time of relapse or progression than at time of diagnosis (P ¼ 0·003), and correlated to CD34 expression and chromosomal abnormalities of leukaemic cells (P ¼ 0·01 and P ¼ 0·001 respectively). The rate and duration of complete remission (CR) did not correlate with the levels of telomerase activity at diagnosis. Among eight patients in first relapse, however, two of three with low levels of telomerase activity re-entered CR, whereas none of five patients with high telomerase activity achieved a second CR. Taken together, telomerase activation/up-regulation in AML is a disease progressionassociated event. Undifferentiated status and chromosomal aberration also lead to the up-regulation of telomerase activity in AML.

The clinical and pharmacokinetic risk factors for toxicity after high-dose methotrexate (MTX) in children with acute lymphoblastic leukemia were evaluated using a multivariate statistical analysis. Plasma samples were collected after 44 24-h infusions of MTX (5 or 8 g/m2) in 13 children (age 3.3-12.9 years) and subsequently analyzed by HPLC to determine the MTX and 7-hydroxymethotrexate (7-OHMTX) concentrations. Toxicity was evaluated according to the WHO criteria. Severe toxicity was not observed. Oral mucositis (WHO grade > or = 1) was significantly related to a high plasma MTX concentration at 28 h after starting the infusion (p = 0.013), a low ratio of plasma 7-OHMTX/MTX at 66 h after starting the infusion (p = 0.049), and a slow clearance of MTX (p = 0.048). The risk of leukopenia (WHO grade > or = 2) increased significantly with the number of courses (p = 0.02). Increasing age and a long exposure to a high MTX concentration in plasma (AUC) were significant risk factors (p = 0.047 and p = 0.009, respectively) for developing elevated liver enzymes (ALAT) (WHO grade > or = 2). This study shows how a statistical model can be used to identify clinical and pharmacokinetic factors that may influence MTX-induced toxicity. The therapeutic ratio could thereby potentially be improved.