The BMI1 polycomb protein represses cyclin G2-induced autophagy to support proliferation in chronic myeloid leukemia cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin's B lymphomas via NF-κB-dependent induction of HIF-1α.

Deregulated expression of glycolytic enzymes contributes not only to the increased energy demands of transformed cells but also has non-glycolytic roles in tumors. However, the contribution of these non-glycolytic functions in tumor progression remains poorly defined. Here, we show that elevated expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but not of other glycolytic enzymes tested, increased aggressiveness and vascularization of non-Hodgkin's lymphoma. Elevated GAPDH expression was found to promote nuclear factor-κB (NF-κB) activation via binding to tumor necrosis factor receptor-associated factor-2 (TRAF2), enhancing the transcription and the activity of hypoxia-inducing factor-1α (HIF-1α). Consistent with this, inactive mutants of GAPDH failed to bind TRAF2, enhance HIF-1 activity or promote lymphomagenesis. Furthermore, elevated expression of gapdh mRNA in biopsies from diffuse large B-cell non-Hodgkin's lymphoma patients correlated with high levels of hif-1α, vegf-a, nfkbia mRNA and CD31 staining. Collectively, these data indicate that deregulated GAPDH expression promotes NF-κB-dependent induction of HIF-1α and has a key role in lymphoma vascularization and aggressiveness.

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production.

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson's disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.

Induction of different types of cell death after normothermic liver ischemia-reperfusion.

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular autophagy, apoptosis, and necrosis. The aim of this study was to investigate these three types of cell death in normothermic liver I-R in rats. A segmental normothermic ischemia of the liver was induced for 120 minutes. Liver autophagy was evaluated by transmission electron microscopy and LC3 (Light Chain 3) immunohistochemical studies. Liver apoptosis was assessed by FLIVO (FLuorescence in vIVO) and TUNEL (TdT-mediated dUTP nick end labeling) assays. Liver necrosis was determined by optical microscopic examination. Autophagy was increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Fluorescence microscopy showed in situ caspase-3 and -7 specific activity to be increased in ischemic liver lobes after 6 hours of reperfusion, compared with nonischemic lobes. Quantitative analysis of apoptotic cells evaluated by the TUNEL method showed a clearly significant increase in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Necrotic cell death was significantly increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes (P < .005). In conclusion, 120 minutes normothermic liver I-R resulted in increased autophagic, apoptotic and necrotic cell death.

Cathepsin B release after imatinib-mediated lysosomal membrane permeabilization triggers BCR-ABL cleavage and elimination of chronic myelogenous leukemia cells.

Imatinib is the leading compound to treat patients with chronic myelogenous leukemia (CML) but the exact mechanism of its anti-leukemic effect is incompletely elucidated. Through inhibition of BCR-ABL, Imatinib blocks several downstream pathways and induces apoptosis of BCR-ABL positive cells. In this study, we analyzed further the mode of action of Imatinib in different appropriate cellular models of CML either sensitive or resistant to Imatinib and in CD34+ cells from CML patients. Pharmacological or short hairpin RNA-mediated inhibition of BCR-ABL triggers lysosomal membrane permeabilization (LMP) that culminates in activation and redistribution of Cathepsin B (CB) into the cytoplasm of CML cells, in which it triggers directly BCR-ABL degradation. Pharmacological inhibition of CB by CA-074Me or small interfering RNA-mediated knock-down of CB partly protects K562 cells from Imatinib-induced cell death and CB overexpression sensitizes these cells to Imatinib killing. Strikingly, Imatinib-triggered LMP, CB activation and BCR-ABL cleavage in CD34+ cells from CML patients and inhibition of CB confers protection against cell death in clonogenic assays of CD34+ primary cells from CML patients. Hence, we describe an original pathway by which Imatinib participates to the elimination of CML cells through LMP and CB-mediated specific degradation of BCR-ABL.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells.

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Liver apoptosis following normothermic ischemia-reperfusion: in vivo evaluation of caspase activity by FLIVO assay in rats.

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular apoptosis. Caspase activation is involved in the initiation and execution of apoptosis. The aim of this study was to determine in vivo caspase activity in normothermic liver I-R in rats. Segmental normothermic ischemia of the liver was induced for 120 minutes in rats. After intravenous injection of the green probe FLIVO, in vivo caspase-3- and -7-specific activity was determined using fluorescence microscopy, in either nonischemic or ischemic liver lobes at 3 and 6 hours after reperfusion. Liver apoptosis was assessed by the deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Fluorescence microscopy showed that in vivo caspase-3- and -7-specific activities were significantly increased (P< .005) in ischemic lobes at 3 and 6 hours of reperfusion, compared with nonischemic liver lobes. Quantitative analysis of apoptotic cells measured by the TUNEL method showed a significant increase among apoptotic cells in ischemic lobes at 3 and 6 hours after reperfusion (P< .005), compared with nonischemic liver lobes. In conclusion, 120-minute normothermic liver I-R resulted in increased caspase-3- and -7-specific activities and in liver cell apoptosis.

Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level.

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition.

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Differential expression of inositol 1,4,5-trisphosphate receptor types 1, 2, and 3 in rat myometrium and endometrium during gestation.

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF(4)(-) was enhanced with advancing gestation. (3)[H]IP(3) binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP(3)R as well as their corresponding proteins, IP(3)R-1, IP(3)R-2, and IP(3)R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP(3)R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.

[Elements of epidemiology and initiation of carcinogenesis in carcinomas of the upper aerodigestive tract. Future therapeutic consequence?]. / Eléments d'épidémiologie et initiation de la cancérogénèse dans les carcinomes des VADS. Conséquence thérapeutique future?

The study of epidemiology and of the carcinogenesis in epidermoid carcinomas of the upper aerodigestive tract shows that their occurrence is not random. Tobacco abuse plays a major role, especially because of benzopyrene, mutagen of the P53 gene, however it is associated with many other potentiating factors: alcohol, metals, hydrocarbures, virus, food, climate, genetic fragility that create genetic lesions at the origin of carcinogenesis. The latter occurs as "field cancerization" with multiple alterations of the mucosa and general attack of the control systems of the differentiation, growth and cell apoptosis which usually protect the cell against the phenomena of carcinogenesis. The P53 protein gene, retinoid receptors as well as the system of detoxifying glutathion S transferase are modified at the very early stage of these diseases, these abnormalities can be logically related to epidemiological data. These data lead us therefore to imagine complementary specific reverting therapies of induced genetic abnormalities, through the reexpression of non mutated gene encoding P53 protein and the use of retinoid. These various modalities are reported hereafter.

Glutathione S-transferase M1 null genotype: lack of association with tumour characteristics and survival in advanced breast cancer.

BACKGROUND: Glutathione S-transferase (GST)M1, a member of the mu class GST gene family, has been shown to be polymorphic because of a partial gene deletion. This results in a failure to express the GSTM1 gene in 50-60% of individuals. Several studies have demonstrated a possible link with the GSTM1-null genotype and susceptibility to cancer. Furthermore, a GSTM1 isoenzyme has been positively associated with protective effect against mutagenic drugs, such as alkylating agents and anthracyclines. OBJECTIVES: To determine whether GSTM1 polymorphisms are associated with tumour characteristics and survival in advanced breast cancer patients, and whether it may constitute a prognostic factor. METHODS: We genotyped 92 patients receiving primary chemotherapy, which included cyclophosphamide, doxorubicine and 5-fluorouracil. The relationships between allelism at GSTM1 and clinicopathological parameters including age, menopausal status, tumour size, grade hormone receptors, involved nodes and p53 gene mutations were analysed. Of the patients with GSTM1-positive genotype, tissue samples obtained before and after treatment were available from 28 cases, allowing RNA extraction and GSTM1 expression by reverse transcription polymerase chain reaction. Relationships with clinical response to chemotherapy, and disease-free and overall survival were also evaluated. The data obtained was analysed using logistic regression to estimate the odds ratio and 95% confidence interval. RESULTS: Of 92 patients, 57.6% (n = 53) were classified as heritably GSTM1-deficient, and 42.4% (n = 39) were of the GSTM1-positive genotype. There were no statistically significant relationships between GSTM1-null genotype and the clinicopathological parameters analysed. No relationship was observed between GSTM1 RNA expression and objective clinical response to chemotherapy. Objective clinical response to chemotherapy was related only to clinical tumour size (P = 0.0177) and to the absence of intraductal carcinoma (P = 0.0013). GSTM1-null genotype had no effect on disease-free or overall survival. The absence of hormone receptors (P = 0.002), the presence of a mutated p53 gene (P = 0.0098) and lack of response to primary chemotherapy (P = 0.0086) were the only factors associated with reduced disease-free or overall survival. CONCLUSIONS: GSTM1-null genotype alone had no effect on tumour characteristics and outcome of patients with advanced breast cancers. The lack of correlation of GSTM1 genotype with clinical tumour features, clinical response to chemotherapy and survival exclude a role for GSTM1 polymorphism as a prognostic factor in advanced breast cancer.

Influence of the fluoroquinolone ofloxacin on the intrinsic expression of multidrug resistance phenotype in HCT-8 human colon carcinoma cells.

The influence of antibiotics, particularly ofloxacin (OF), a commonly used antimicrobial fluoroquinolone, on the multidrug resistance (MDR) phenotype of the HCT-8 cell line was studied. This cell line was grown in OF containing medium for several months and the expression of the MDR phenotype was followed through the analysis of the expression and functionality of the P-glycoprotein (Pgp), the chemosensitivity to daunorubicin (DNR), and the mRNA expression of mdr-1, multidrug resistance-associated protein (MRP), and topoisomerase IIalpha and IIbeta genes. Replacement of OF by penicillin streptomycin (PS) resulted in a significant decrease in mdr-1 mRNA expression, which was found to correlate with a decrease in the expression and functionality of the Pgp. After antibiotic starvation for 4 weeks, cells grown in antibiotic-free medium were then exposed to PS or OF; these cells showed an increase in mdr-1 mRNA/Pgp and MRP mRNA expression without a decrease in DNR cytotoxicity. OF cultured cells exhibited a significant increase in Pgp expression without evidence of the functionality of the Pgp. An increase in topoisomerase IIalpha mRNA expression was observed with time and with the number of passages of the cell line without any relationship to the presence of antibiotics in the culture medium. These results showed that extensive use of antibiotics, particularly the quinolones, can modify the phenotype of the HCT-8 colon adenocarcinoma cell line.

Influence of SDZ-PSC833 on daunorubicin intracellular accumulation in bone marrow specimens from patients with acute myeloid leukaemia.

The multidrug resistance (MDR) modulating activity of SDZ-PSC833 (PSC), a non-immunosuppressive cyclosporine analogue, was investigated and compared with cyclosporin A (CSA) in bone marrow clinical specimens from 45 patients with acute myeloid leukaemia (AML) taken at diagnosis, using double-labelling flow cytometry with simultaneous determination of P-glycoprotein (PGP) expression and intracellular daunorubicin fluorescence (IDF). On the basis of pre-clinical results in multidrug-resistant K562 leukaemic cells, concentrations leading to iso-effective complete restoration of IDF were used: 5 and 10 micromol/l, respectively for PSC and CSA. In the clinical specimens, PGP expression was correlated with a significant decrease in IDE PSC was found to be significantly more potent than CSA since it was found to induce a significant increase in IDF in a higher number of cases and to a higher extent than CSA. PGP-unrelated activity of PSC was also observed in specimens expressing no PGP but exhibiting low IDF, thus probably expressing alternative resistance mechanisms. The results confirm the potency of PSC as MDR-modulating agent in clinical AML specimens whose resistance pattern differed from that of highly resistant cell models and suggest that the activity of PSC is not limited to P-glycoprotein inhibition.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

Distribution of transforming growth factor beta1-binding proteins and low-affinity receptors during odontoblast differentiation in the mouse.

Transforming growth factor-beta1 (TGF-beta1) was immunolocalized within differentiated odontoblasts and ameloblasts while LAP-beta1 was detected at the apicol pole of odonotoblasts and ameloblasts and in predentine. Anti-LAP-beta1 antibodies also stained the epithelial-mesenchymal junction (EMJ). Decorin was immunolocalized in young functional odonotoblasts and in both predentine and dentine. Biglycan was similarly distributed but absent from dentine. Immunostaining with anti-latent TGF-beta1 binding protein-1 (LTBP-1) showed fibrillar structures located at the EMJ and between predontoblasts and odontoblasts; at older states staining was restricted to the dental papilla and sac. Thus differentiated odonotoblasts express TGF-beta1 and in a more restricted manner decorin, biglycan and LAP-beta1; it can be assumed that TGF-beta1 is able to interact with the three molecules present in predentine. Earlier, LTBP-1 and LAP-beta1, both present at the EMJ, may contribute to odontoblast differentiation.