Search PubMed⌕ Search

Biomedical subjects

D Seigneurin

Publications and source records attributed to D Seigneurin.

At least 19 recordsLinked to original sources
PubMed2001

Dag-1 carcinoma cell in studying the mechanisms of progression and therapeutic resistance in bladder cancer.

OBJECTIVE: We describe a new human bladder carcinoma cell line (DAG-1) established from a resected bladder cancer fragment and maintained in culture for more than 5 years and over 300 passages. METHODS AND RESULTS: Immunological, biochemical and molecular analysis showed that the DAG-1 cells (62 chromosomes) express the cytokeratines 8, 13, 18 and 20 that confirm their epithelial origin as well as numerous cytokine and cytokine receptor mRNAs. They secrete tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitors (PAI-1 and PAI-2), and express u-PA receptors (u-PAR/CD87) at their surface. DAG-1 cells are resistant to TNFalpha- and IFNgamma-induced apoptosis, two cytokines secreted in the urine of Calmette-Guérin bacillus-treated patients and involved in the tumor regression. CONCLUSION: The DAG-1 cell line is a useful tool, both in vitro and in vivo, to study the progression of bladder tumors and their mechanisms of resistance to immunotherapy in relation with PAI-2 and antioxidant enzymes.

Aged↗
PubMed2000

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Expression of E-, P-, n-cadherins and catenins in human bladder carcinoma cell lines.

PURPOSE: Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classical cadherins, E-, P- and N-cadherins, are known to self-associate from their extracellular domain, while their cytoplasmic domain interacts with either beta-catenin or plakoglobin (gamma-catenin), which in turn is bound to alpha-catenin that links the complex to the actin cytoskeleton. The aim of the present study was to analyze the expression of E-, P- and N-cadherins and catenins in human bladder carcinoma cells. MATERIALS AND METHODS: Five human bladder carcinoma cell lines, representing a variety of differentiation states, were grown in cell culture. We performed a cell aggregation assay, specific for biological cadherin activity. The expression of cadherins and catenins was analyzed by immunocytochemistry, Western blotting and RT-PCR. The interactions between cadherins and catenins were assessed by immunoprecipitation. RESULTS: We observed a reduced E-cadherin expression in the poorly differentiated and invasive-tumor derived cells. Interestingly, immunofluorescence study reveals the persistent localization of catenins at intercellular contacts in two E-cadherin deficient cell lines (T24 and TCCSUP) which yet exhibit an epithelial-like morphology and a calcium-dependent adhesive capacity. This suggests that other cadherin(s) are expressed in these both cell lines. P-cadherin, another epithelial cadherin, is expressed only in E-cadherin positive cells. On the other hand, N-cadherin is present at cell-cell borders in the very anaplastic cell lines, T24 and TCCSUP, and is able to link beta-catenin or plakoglobin. CONCLUSION: These results indicate that N-cadherin may participate in intercellular adhesion, while facilitating bladder tumorigenesis.

Actins↗
PubMed2000

[Breast, cervical and colo-rectal simultaneous mass screening program for women 50 to 69 years old in Isere].

A cervical screening campaign is implemented in the Isere department since 1990 for women aged 50 to 69, together with breast cancer and colo-rectal cancer screening. The attendance rate is about 30% but a survey performed among this population shows that 68.6% of these women did presented for screening during the year following their invitation. One and a half per cent of all smears were abnormal or ASCUS smears. Cancer detection rate for invasive cancer and CIN III was 1.3/1000. General practitioners and gynaecologists took the same part in the programme. During a postal survey conducted among them, they declared that they felt concerned with cancer screening, even if they met some difficulties. This results suggest that nominative invitations and a good cooperation of GPs may improve the coverage of eligible women. For the future success of the National Programme, which is to be implemented in France, organisational arrangements have to be set up. The review of positive points and difficulties met by Isere's local programme may help to discuss it.

Aged↗
PubMed2000

Nucleus labeling or membrane labeling for studying the proliferation of drug treated cells?

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia (AML). This study compared cell cycle analysis (nuclear labeling) with cell division analysis (membrane labeling, PKH67) for studying the proliferation of cells cultured with daunorubicin (DNR) and/or Cytarabine (Ara-C), drugs commonly used in AML treatment. PKHs are a family of lipophilic, fluorescent membrane intercalating dyes. When labeled cells divide, the resulting daughter cells receive half the label, reducing fluorescence intensity to one-half that of the parent cells. DNR has the disadvantage of overlapping the spectrum of propidium iodide (PI), which is the most commonly used marker of membrane integrity. In this study, necrosis was evaluated using TOTO-3, a marker of nucleic acids emitting fluorescence above 645 nm and which incorporates cells with disrupted membranes. Comparison of cell cycle analysis with cell division for studying proliferation revealed that PKH67 was a marker of choice for analyzing the mitotic process in cells cultured with drugs.

Acute Disease↗
PubMed2000

[Cytometry].

Explore the source record for details and available documents.

Cytological Techniques↗
PubMed1999

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines.

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.

Antibiotics, Antineoplastic↗
PubMed1999

Chromosome 11 aneuploidy detected by fluorescent in situ hybridization (FISH) in breast cancer: relation with progesterone receptor expression.

Progesterone receptor (PR) expression is known to be impaired in breast cancer. As the PR gene is located on chromosome 11 which is also often affected, we studied their relationship in 15 patients with breast carcinoma. Tumoural imprints were used for PR immunocytochemistry and for FISH with chromosome 11 centromeric probes. Distribution profiles of chromosome 11 number in PR+ and PR- cell populations were examined. No difference in the number of chromosome 11 was found between PR+ and PR- breast tumours. Thus, loss of PR expression in breast cancer cannot be explained only by loss of chromosome 11; other genetic or non-genetic mechanisms should be advanced.

Aneuploidy↗
PubMed1998

Usefulness of PKHs for studying cell proliferation.

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies. (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.

Biomarkers↗
PubMed1998

[Cytology of fluids of articular puncture].

Cytologic examination of synovial fluid has been shown to be useful in the diagnosis of various joint diseases. Few pathologists are proficient in the interpretation of the occasional synovial fluids that most cytology laboratories receive. The majority of the aspirated synovial fluids are sent only for bacterial culture and valuable information from the cytologic examination is missed. We describe how pathologists should perform analysis of these fluids and what kind of information they can provide to the physician.

Cytodiagnosis↗
PubMed1997

Expression of three cell adhesion molecules in bladder carcinomas: correlation with pathological features.

Recently, independent studies have shown that the expression of two integrin chains, beta 4 and alpha 2, plus the epithelial cadherin are related to tumour progression in human bladder carcinomas. For the first time, we compare the expression of these three cell adhesion molecules using immunohistochemical analysis of consecutive cryosections from a series of 50 bladder tumors. E-cadherin, beta 4, and alpha 2 were strongly expressed in normal urothelium. A majority of non-invasive bladder cancers stained positively for E-cadherin (62%), whereas only 29% expressed normal positivity for alpha 2, and only 35% for beta 4. However, most invasive tumours presented an aberrant expression of alpha 2 (81%), beta 4 (100%), and E-cadherin (75%). We studied the correlation of immunoreactivity with histological grade and stage. The alpha 2 pattern was not correlated with stage and grade. In contrast, loss of normal beta 4 expression was significantly related to increasing tumour grade and deep invasion with a higher correlation for grade. Finally, E-cadherin expression was highly correlated with stage, but not with grade. Thus our results indicate that, although many invasive bladder tumours presented a disorder in expression of the two integrins alpha 2 and beta 4, E-cadherin appeared to be a better market of invasiveness in bladder carcinomas.

Adult↗
PubMed1997

Limits of flow-cytometry histogram analysis methods to assess bladder tumour antigen expression.

Tumour-associated antigens detected in cells obtained from bladder washings or tumours are useful markers in bladder cancer. Flow cytometry is commonly used to quantify immuno-stained cells. A straightforward way to analyze data is to count the fluorescent cells above a threshold empirically determined on a control histogram representation. However, specific antigens expressed at highly variable rates give rise to wide range distributions in flow cytometry as illustrated when a mucin antigen for urinary bladder was titrated by M344 monoclonal antibody in urothelial cancer cells. We have evaluated several methods of background estimation and subtraction in order to determine the proportion of M344 Mab positive cells. These include threshold setting (Histogram Shape Dependent (HSD) threshold developed in this study, 2% preset or 5% preset background), subtraction of the blank from the test histograms, and Kolmogorov-Smirnov statistical test. The HSD method appeared to be a more reliable method for background estimation; however, in the case of very low antigen expression, where specific fluorescence histograms could hardly be distinguished from that of the background, fluorescence microscopy remained the only valid method, since it allowed the distinction between specific and non-specific fluorescence on the basis of structural differences between the two.

Antibodies, Monoclonal↗
PubMed1997

Proliferation of LAMA-84 and LAMA-87 cell lines is modulated by autocrine loops involving M-CSF and TGF-beta.

The erythromegakaryocytic cell line (LAMA-84) and the erythroeosinophilic cell line (LAMA-87) were used to study receptor expression and receptor-mediated response to monocyte/macrophage colony-stimulating factor (M-CSF) and transforming growth factor beta (TGF-beta), two modulators of cell proliferation. As demonstrated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), c-fms and M-CSF mRNA were expressed in both cell lines. M-CSF was detected in the supernatant of both cell lines and addition of a neutralizing anti-M-CSF antibody inhibited cell growth. The two LAMA cell lines were found to express TGF-beta1, -beta2, and -beta3 mRNAs and to secrete TGF-beta mostly in latent form. Addition of anti-TGF-beta antibodies to the culture medium increased their proliferation, whereas TGF-beta1 inhibited cell proliferation by downregulating the c-myc mRNA. These results show that the proliferation of both LAMA cell lines is positively and negatively regulated by autocrine mechanisms, implying the presence of M-CSF and TGF-beta, respectively. They suggest that similar autocrine loops could be involved in the growth regulation of leukemic cells in vivo.

Animals↗
PubMed1997

Expression of E-cadherin and alpha-,beta- and gamma-catenins in human bladder carcinomas: are they good prognostic factors?

E-cadherin, the epithelium-specific cadherin, is known to play a major role in tumor progression in many human carcinomas, via intercellular homophilic Ca2+-dependent adhesion. This adhesion is mediated by a group of cytoplasmic proteins, including the alpha-, beta- and gamma-catenins that link the E-cadherin to the actin cytoskeleton. Recent studies have shown that loss or reduction of either E-cadherin or catenin expression was strictly related to clinicopathological data in bladder tumors, and E-cadherin might constitute prognostic factors in bladder carcinogenesis. Here we continued a preliminary work on E-cadherin in bladder cancer. In an effort to evaluate their possible prognostic value, we investigated both E-cadherin and catenins in 99 bladder tumors by immunohistochemistry. E-cadherin and all the catenins were strongly expressed in normal urothelium. Regarding histopathological data, the tumors examined showed that the disrupted expression of each molecule, except for gamma-catenin, was directly related to increasing tumor grade (mainly for alpha- and beta-catenin) and deep invasion (p < or = 0.01). The aberrant expression of E-cadherin and beta-catenin was also correlated to the presence of distant metastasis (p < 0.05). However, only abnormal expression of a-catenin was associated with poor survival (p = 0.037). Therefore our results suggest that alpha-catenin is directly involved in tumor invasion and dedifferentiation and is the only protein of any prognostic value, albeit low in patients with bladder cancer.

Adult↗
PubMed1996

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Cell Division↗
PubMed1996

Prognostic value of quantitative cytometry in a series of 415 T1T2/N0N1/M0 breast cancer patients--preliminary results.

Identifying prognostic markers in local regional breast carcinomas remains an important challenge today. DNA content obtained by flow cytometry, has been found to be of prognostic value; results with other methods remain less clear. This report describes DNA image cytometry patterns which are assessed with respect to disease-free survival. From June 1982 to December 1992, 415 patients under 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), N0 (65.1%) N1 (34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75) and 58.8% of the patients were premenopausal; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade and, oestrogen receptor content were used in decision-making for adjuvant treatment: hormonotherapy (48%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was carried out on air dried smears to be analysed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index; DNA histogram, integrated optical density, DNA malignancy grade, ploidy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (IOD) (P = 0.018) and DNA malignancy grade (P = 0.017) had a significant prognostic value on disease-free survival estimated by the Kaplan-Meier method. When these parameters were included in a Cox proportional regression hazards model, PR (P = 0.01), Scarff-Bloom histological grading (P = 0.02), axillary clearance (P = 0.04) were significant; however, in the same model, taking into account the axillary lymph node histological status, IOD was significant for pN- patients (P = 0.03), and proliferation index (P = 0.03) was significant for pN+. Such results need to be updated with a longer median follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), should be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

Adult↗
PubMed1996

Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells.

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.

Antigens, Nuclear↗