Genetic factors (VKORC1, CYP2C9, EPHX1, and CYP4F2) are predictor variables for warfarin response in very elderly, frail inpatients.

Determining the optimal dose of warfarin for frail elderly patients is a challenging task because of the low dose requirements in such patients, the wide interindividual variability of response, and the associated risk of bleeding. The objective of this study was to address the influence of 13 common variations in eight genes on the maintenance dose of warfarin in a cohort of frail elderly inpatients. For our study, we enrolled 300 Caucasian subjects who were hospital inpatients, with a mean age of 86.7 +/- 6 years. In addition to age, genetic variants of VKORC1, CYP2C9, CYP4F2, and EPHX1 were found to be significant predictor variables for the maintenance dose of warfarin, explaining 26.6% of dose variability. Among 132 patients in whom warfarin therapy was initiated with the same low-dose regimen, we studied the relative influences of genetic and nongenetic factors. The time to first international normalized ratio (INR) > or =2 was influenced by VKORC1 and CYP2C9 genotypes (P = 0.0003 and P = 0.0016, respectively); individuals with multiple variant alleles were at highest risk for overanticoagulation (INR >4) (odds ratio, 12.8; 95% confidence interval, 2.73-60.0). In this special population of frail elderly patients with multiple comorbidities and polypharmacy, we demonstrated the main impact of genetic factors on warfarin response.

Clinicopathological significance of mitochondrial D-Loop mutations in head and neck carcinoma.

Mitochondrial DNA mutations have been reported in several types of tumours, including head and neck squamous cell carcinoma (HNSCC). The noncoding region of the Displacement-Loop (D-Loop) has emerged as a mutational hotspot and we recently found that they were associated with prognosis and response to 5 fluorouracil (5FU) in colon cancers. In order to evaluate the frequence of D-Loop mutations in a large series of HNSCC and establish correlations with clinicopathologic parameters, we sequenced the D-Loop of 109 HNSCC before a treatment by neoadjuvant 5FU-cisplatin-based chemotherapy and surgery. Then, we correlated these mutations with prognosis and response to chemotherapy. A D-Loop mutation was identified in 21% of the tumors, the majority of them were located in a C-tract (D310). The prevalence of D310 mutations increased significantly with the number of cytosines in the matched normal tissue sequence (P=0.02). Hypopharyngeal cancer was significantly more frequent (P=0.03) and tobacco consumption more important (P=0.01) in the group of patients with D-Loop mutation. The presence of D-Loop mutation was not associated with prognosis or with response to neoadjuvant chemotherapy. These results suggest that D-Loop mutations should be considered as a cancer biomarker that may be useful for the early detection of HNSCC in individuals at risk of this cancer.

p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series.

PURPOSE: The tumor suppressor gene p53 plays a crucial role in cell cycle control and apoptosis in response to DNA damages. p53 gene mutations and allelic losses at 17p are one of the most common genetic alterations in primary head and neck squamous cell carcinoma (HNSCC). Alterations of the p53 gene have been shown to contribute to carcinogenesis and drug resistance. PATIENTS AND METHODS: In this prospective series, patients with HNSCC were treated with cisplatin-fluorouracil neoadjuvant chemotherapy. p53 status was characterized in 106 patients with HNSCC (p53 mutations, allelic losses at p53 locus, and plasma anti-p53 antibodies) to determine the existence of a relationship between p53 gene status and response to neoadjuvant chemotherapy. RESULTS: Exons 4 to 9 of the p53 gene were analyzed, and mutations were found in 72 of 106 patients with HNSCC. p53 mutations were associated with loss of heterozygosity at chromosome 17p (P <.001). The prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than in the group of responders (81% v 61%, respectively; P <.04). When compiling p53 mutations and anti-p53 antibodies in plasma, the correlation between p53 status and response to chemotherapy was significant (87% v 57%, respectively; P =.003). A multivariate analysis showed that p53 status is an independent predictive factor of response to chemotherapy. CONCLUSION: This prospective study suggests that p53 status may be a useful indicator of response to neoadjuvant chemotherapy in HNSCC.

A protein kinase C inhibitory activity is present in the human T lymphoblast cell line Jurkat.

The human lymphoblast cell line Jurkat is widely used as a model system for studying signal transduction pathways during lymphocyte activation. We report the presence of a potent endogenous inhibitor of protein kinase C (PKC) in the cytosolic fraction of Jurkat cells. This inhibitor is not diffusible and is thermolabile; it is assumed to be a protein. It was separated from PKC by ion-exchange chromatography on DEAE-cellulose. The inhibitory activity was partially reversed by increasing the concentration of the PKC substrate; increasing that of PKC activators (calcium and phospholipids) was without effect. PKC activity was inhibited by more than 90% in the crude cytosolic fraction but the inhibition could be completely reversed by diluting the cell extract. This inhibitory activity could not be detected in the cytosol from normal lymphocytes or from lymphoblasts from leukemic patients.

Retinoic acid inhibits the expression of cytidine deaminase linked to the differentiation of the human leukemic cell line HL-60.

The activity of cytidine deaminase markedly increases during the differentiation of HL-60 cells induced by dimethylsulfoxide or 1,25-dihydroxy vitamin D3, but does not increase when the inducer is retinoic acid. Here it is demonstrated that retinoic acid inhibits the increase in cytidine deaminase activity elicited by the other two inducers. This inhibitory effect of retinoic acid (i) was not the result of a direct action on the enzymatic activity; (ii) was correlated with the differentiating effect of retinoic acid, as indicated by the similar time-course and dose-dependence of both effects, and by additional studies with various retinoids and with an HL-60 variant resistant to retinoic acid-induced differentiation; (iii) required the continued presence of the drug for more than 24 h, and could not be reversed after 48 h; (iv) was manifest, after a lag-time of 24 h, at whatever time retinoic acid was added during the 5 days of treatment of the cells with the differentiation inducers; and (v) was prevented by the addition of the protein synthesis inhibitor cycloheximide. These data indicate that retinoic acid negatively regulates the expression of cytidine deaminase in HL-60 cells, and suggest that this effect is mediated by a protein, the synthesis of which should be controlled by the nuclear receptor of retinoic acid.

Influence of adenosine deaminase inhibition on the phosphoinositide turnover in the initial stages of human T cell activation.

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'-deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase-deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P-prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P-loading period. However, when the cells were stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP-containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells.

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

Synergistic toxicity of pyrazofurin and cytidine in cytidine deaminase deficient lymphoid cells (Raji).

The intermediary metabolism of pyrimidine nucleosides was studied in a line of human B lymphoblasts (Raji) in which pyrimidine de novo synthesis deficiency was pharmacologically induced by pyrazofurin. It was found that Raji cells are cytidine deaminase deficient that cytidine has a synergistic effect on the toxicity of pyrazofurin towards these cytidine deaminase deficient cells, affecting both the proliferation and the viability of the cells. Indirect evidences suggest that this synergistic toxicity is not mediated by an effect on nucleoside diphosphate reductase nor on the first steps of pyrimidine de novo synthesis.

Cytidine deaminase activity of human normal and malignant lymphoid cells.

Cytidine deaminase activity was determined by a radioisotopic assay in extracts of peripheral blood mononuclear cells of normal individuals and of patients with acute lymphoblastic leukaemia. The normal enzyme activity had a broad pH optimum between pH 6.5 and 8.0; apparent Km values for cytidine and deoxycytidine were 3.6 +/- 0.6 mumol/l and 26.5 +/- 3.5 mumol/l, respectively; the activity was resistant to heat inactivation; of the various effectors tested, only uridine, deoxyuridine and tetrahydroxyuridine had inhibitory effects. Cytidine deaminase activity was markedly decreased in lymphoblasts of patients with acute lymphoblastic leukaemia; enzyme activity was related to the percentage of circulating blast cells, and not to the clinical, cytological or immunological characters of the leukaemia.

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis.

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 microM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 microM. Guanine, guanosine and 2'-deoxyguanosine at concentrations up to 500 microM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 microM adenine, adenosine, 2'-deoxyadenosine or 100 microM 2'-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2'-deoxyguanosine toxicity was dependent and 2'-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.