Comparative evaluation by semiquantitative reverse transcriptase polymerase chain reaction of MDR1, MRP and GSTp gene expression in breast carcinomas.

Identification and quantitative evaluation of drug resistance markers are essential to assess the impact of multidrug resistance (MDR) in clinical oncology. The MDR1 gene confers pleiotropic drug resistance in tumour cells, but other molecular mechanisms are also involved in drug resistance. In particular, the clinical pattern of expression of the other MDR-related genes is unclear and their interrelationships are still unknown. Here, we report standardization of the procedures used to determine a reliable method of semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using a standard series of drug-sensitive and increasingly resistant cell lines to evaluate the expression of three MDR-related genes, i.e. MDR1 (multidrug resistance gene 1), MRP (multidrug resistance related protein) and GSTp (glutathione-S-transferase p), reported to be endogenous standard genes for normalization of mRNAs. A total of 74 breast cancer surgical biopsies, obtained before any treatment, were evaluated by this method. When compared with classical clinical and laboratory findings, GSTp mRNA level was higher in diploid tumours. However, the main finding of our study suggests a clear relationship between two of these MDR-related gene expressions, namely GSTp and MRP. This finding provides new insight into human breast tumours, which may possibly be linked to the glutathione conjugate carrier function of MRP. Well defined semiquantitative RT-PCR procedures can therefore constitute a powerful tool to investigate MDR phenotype at mRNA levels of different related genes in small and precious tumour biopsy specimens.

Cisplatin-induced apoptosis and p53 gene status in a cisplatin-resistant human ovarian carcinoma cell line.

Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.

Nasal polyposis pathogenesis: a flow cytometric and immunohistochemical study of epithelial cell proliferation.

In nasal polyps, constantly associated with chronic inflammation, frequent epithelial morphological changes (squamous metaplasia, secretory hyperplasia) suggest a dysregulation of epithelial cell proliferation. Cell proliferation in nasal respiratory epithelium was therefore evaluated in nasal polyposis. In 20 patients, we compared cell proliferation in mucosa from the inferior turbinate to these in nasal polyps using two methods: Flow cytometry analyzing first the ploidy and the percentage of S-phase cells (propidium iodide DNA labeling), secondly the percentage of Ki-67-labeled cells and the green fluorescent index (fluorescein-conjugated anti-human Ki-67 antigen labeling, and thirdly the percentage of Ki-67-labeled cells being in S-phase. Immunohistochemistry, quantifying the expression of Ki-67 antigen in the epithelium permitting to calculate a Ki-67 index. All cell-populations studied were diploid. Percentages of S-phase cells, Ki-67-labeled cells, Ki-67 labeled cells being in the S-phase and green fluorescence index was significantly higher in nasal polyps than in mucosa Ki-67 index were significantly higher in nasal polyps than in mucosa in the epithelium. Epithelial cell proliferation which is therefore increased in nasal polyp could play an important role in nasal polyposis pathogenesis and its relationships with inflammation can be suggested.

Increased epithelial cell proliferation in nasal polyps.

OBJECTIVE: To detect, quantify, and compare respiratory epithelial cell proliferation in nasal mucosa and polyps from patients with nasal polyposis. DESIGN: Cohort study. SETTING: Patients and samples were selected at the Hôpital Intercommunal de Créteil (France). Flow cytofluorometry and immunohistochemistry were performed at Hôpitaux Tenon and Mondor (Université Paris [France] VI et XII). PATIENTS: Twenty-one patients undergoing endoscopic ethmoidectomy for treatment of nasal polyposis. METHODS: In 10 cases, epithelial cells were removed from frozen inferior turbinate mucosa and polyps by mechanical disaggregation and were then analyzed by flow cytofluorometry, providing the cell DNA content (propidium iodide labeling) and the percentage of S-phase cells. In 11 cases, inferior turbinate mucosa and polyps were fixed in formaldehyde and embedded in paraffin. Proliferating cell nuclear antigen expression in the epithelium was quantified by immunohistochemistry; a proliferating cell nuclear antigen index was calculated for each sample in the basal area, suprabasal area, and full height of the epithelium. RESULTS: All cell populations studied were diploid, and percentages of S-phrase cells were significantly higher in nasal polyps than in mucosa. Proliferating cell nuclear antigen indexes were significantly higher in nasal polyps than in the suprabasal area and full height of the mucosal epithelium. CONCLUSION: Cell proliferation is increased in epithelium from nasal polyps. Epithelial damage caused by inflammatory mediators could induce this increased cell proliferation via epithelial repair processes. Inflammatory cells could up-regulate epithelial cell proliferation by secreting growth factors.

[Flow cytometry in the study of meningiomas. Preliminary results and attempt at clinical correlation]. / Apport de la cytométrie en flux dans l'étude des méningiomes. Résultats préliminaires et essai de corrélation clinique.

Meningiomas are meningeal primitive tumors. These benign neoplasms can recur but the rate of recurrence is unknown as there is no reliable factor of predictibility. The aim of this study was to test Flow Cytometry based on clinical data and follow up in a series of meningiomas operated on in a neurosurgical department. This method allows the study of DNA content matched with the study of the cellular cycle. S cellular phase was chosen to be tested related to immunostaining with 2 proliferating markers, Ki67 and PCNA. This prospective study was carried out on neurosurgical samples, immediately frozen. On the one hand, results confirm well known discrepancies between Ki67 and PCNA immunostainings. On the other hand, two recurrent meningiomas belong to the diploid group. This is unexpected as aneuploid tumors are known to be biologically more agressive than diploid tumors.

[In vitro liberation of lymphocytic factors inhibiting migration of human leukocytes induced by gold, cadmium and mercury chlorides]. / Libération, in vitro, de facteurs lymphocytaires inhibant la migration des leucocytes humains, induite par les chlorures d'or, de cadmium et de mercure.

The presence of 300 microgram gold and cadmium and 10 microgram mercury chloride per millitre of survival medium, during the process of T.I.M.L., results in the release by human lymphocytes of soluble factors which inhibit migration of leucocytes (L.I.F. and M.I.F.). This stimulation of cellular immunity suggests that the anti-infectious therapeutic effect of gold and mercury inorganic salts results from activation of the histiocytary system, through the release of these lymphokins.