Biomedical subjects

V Pierrefite

Publications and source records attributed to V Pierrefite.

7 recordsLinked to original sources

PubMed1999

Dihydropyrimidine dehydrogenase deficiency and fluorouracil-related toxicity.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of 5-fluorouracil (5-FU) catabolism. We report lymphocytic DPD data concerning a group of 53 patients (23 men, 30 women, mean age 58, range 36-73), treated by 5-FU-based chemotherapy in different French institutions and who developed unanticipated 5-FU-related toxicity. Lymphocyte samples (standard collection procedure) were sent to us for DPD determination (biochemical method). Among the whole group of 53 patients, 19 had a significant DPD deficiency (DD; below 150 fmol min(-1) mg(-1) protein, i.e. less than 70% of the mean value observed from previous population study). There was a greater majority of women in the DD group (15 out of 19, 79%) compared with the remaining 34 patients (15 out of 34, 44%, P<0.014). Toxicity was often severe, leading to patient death in two cases (both women). The toxicity score (sum of WHO grading, theoretical range 0-20) was twice as high in patients with marked DD (below 100 pmol min(-1) mg(-1) protein, n = 11, mean score = 13.2) compared with patients with moderate DD (between 150 and 100 pmol min(-1) mg(-1) protein, n = 8, mean score = 6.8), P = 0.008. In the DD group, there was a high frequency of neurotoxic syndromes (7 out of 19, 37%). The two deceased patients both had severe neurotoxicity. The occurrence of cardiac toxicity was relatively rare (1 out of 19, 5%). These data suggest that women are particularly prone to DPD deficiency and allow a more precise definition of the DD toxicity profile.

Adult↗

PubMed1998

A clinically applicable assay for tumoral thymidylate synthase combining reverse transcriptase-PCR and high-performance liquid chromatography.

The main clinically relevant cellular target of 5-fluorouracil (5-FU) is the enzyme thymidylate synthase (TS). Both preclinical data and clinical data in digestive tract cancer indicate that an increased amount of TS in tumours can predict for 5-FU resistance. We developed an automated method combining the principle of RT-PCR coupled with HPLC separation and quantification. The RT-PCR HPLC method was applied to TS determination in tumoral biopsies from patients with colorectal cancer. The PCR samples were separated and quantified using a polystyrene divinylbenzene C 18 column. Within 22 min, it was possible to elute 18 peaks representing DNA sizes ranging from 34 to 622 bp. Both separation and quantification of beta 2 microglobulin (beta 2m, internal standard) and TS PCR products were achieved in approximately 10 min per sample. Validation of the RT-PCR HPLC method was established by comparing RT-PCR quantification of TS after electrophoresis and HPLC and by comparing the RT-PCR quantification of TS after HPLC with the classical biochemical method. The proposed HPLC method offers a 10-50 fold sensitivity advantage over electrophoresis. In addition, this RT-PCR HPLC procedure allows not only the quantification of TS expression but also the direct collection of unaltered amplified DNA sequence which could be useful for sequencing analysis, since TS mutations have been described. The present RT-PCR HPLC method for determining TS expression in tumoral biopsies is a valuable analytical approach as it is specific, sensitive and clinically applicable.

Adult↗

PubMed1998

Treatment of hepatic metastases of colorectal cancer by electrochemotherapy: an experimental study in the rat.

BACKGROUND: Electrochemotherapy, which consist of local or systemic administration of a cytotoxic agent followed by application of electric pulses to a tumor, has proved effective for various types of tumors in animals and for cutaneous and head and neck cancers in human beings. This is the first study to investigate the efficacy of electrochemotherapy for treatment of hepatic metastases of colorectal cancer in the rat. METHODS: After induction of a solitary hepatic metastasis in 36 male BDIX rats, the animals were randomized to one of four groups: B-E-(no treatment), B+E-(intratumoral bleomycin), B-E+ (application of electric pulses to the tumor), and B+E+ (electrochemotherapy: intratumoral bleomycin followed by application of electric pulses). RESULTS: Groups B-E and B-E+ had no tumor response. Group B+E had one partial response. Group B+E+ had seven partial responses and two complete responses. The difference in terms of response between group B+E+ and the other three groups was statistically significant (P < .05). Comparison of the mean posttherapy tumor volumes (B-E-, 50.6 mm3; B+E-, 58.7 mm3; B-E+, 46 mm3; and B+E+, 5.65 mm3) revealed a significantly smaller residual tumor in group B+E+ than in the other three groups (P < .05). CONCLUSIONS: Electrochemotherapy is an effective means to reduce the volume of hepatic metastases of colorectal cancer in the rat. Additional research is required to determine the optimum treatment duration, dose effects, volume of tumor that can be treated by electrochemotherapy, and impact on survival. Such experimental studies are indispensable prerequisites for clinical trials.

Animals↗

PubMed1997

Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method.

Retinoic acid receptor beta (RAR-beta) seems to be a useful intermediate marker in trials of retinoids, and the aim of this work was to describe a fast, sensitive, and routine applicable method to measure RAR-beta expression in tumor biopsies. We developed a new technique combining reverse transcription-PCR with a colorimetric ELISA detection of amplification products. The principle of this nonradioactive method is based on digoxigenin labeling of PCR products during amplification. Amplified DNA is hybridized with a biotinylated capture probe. The generated hybrid is immobilized on a streptavidin-coated microtiter plate, and detection is performed with the use of an antidigoxigenin peroxidase conjugate. We applied this method to quantify the expression of RAR-beta and an internal control (beta2 microglobulin) in laryngeal tumors. We found a detection threshold at 50 pg of PCR products, which represents a 100-fold improvement when compared to the detection limit of ethidium bromide detection. The method was reproducible (intra- and interassay reproducibilities at 7 and 5%, respectively). We used this technique for determining RAR-beta expression in 20 patients with laryngeal carcinoma and in 20 patients without cancer. The data show that the value of the RAR-beta:beta2 microglobulin ratio is decreased in tumoral versus nontumoral specimens (P = 0.0012), which is consistent with previously published results.

Adult↗

PubMed1996

Conditional requirement for sequences distinct from the replication origin during episomal establishment of the BPV1 genome.

Removal from BPV1 DNA of a short segment (nt. 4786-5045) that contains several protein binding sites and is required for efficient replication in short term assays prevents its autonomous maintenance in cell lines established by selection in G418 medium after cotransfer of neo(r). In contrast, transformed cell lines established from foci, which express the viral genes at higher levels, maintain extrachromosomal copies of the deleted DNA. Two modes of maintenance of the viral genome are thus distinguished by their requirement for sequences in this region.

Animals↗

PubMed1995

Replication efficiency of bovine papillomavirus type 1 DNA depends on cis-acting sequences distinct from the replication origin.

The viral elements required for the initiation of replication of bovine papillomavirus type 1 DNA include the origin region and two trans-acting factors, the E1 and E2 proteins. We now report that the replication efficiency of a DNA molecule which contains these three elements is modulated by other viral sequences. By measuring the extent of replication of deleted viral genomes in transfected mouse cells, we identified sequences required for maximal efficiency. Addition of these sequences to a construct carrying only the minimal origin region increased its replication. Among these cis-active elements, we identified a 69-bp fragment (nucleotides 4921 to 4990) which contains at least two binding sites for cellular proteins. One of them is the murine protein termed CDEBP, which recognizes the octameric motif ATCACGTG, identical to the yeast CDEI element. Either deletions affecting this CDEI box or a point mutation which impairs binding of CDEBP markedly decreased the extent of viral DNA replication. They had no detectable effect on viral transcription.

Animals↗

PubMed1992

Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast.

We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1). Such constructs could constitute convenient shuttle vectors between yeast and mouse cells. However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae. A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus. One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus. Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment. This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element. Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent. These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.

Animals↗