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Biomedical subjects

M P Merville

Publications and source records attributed to M P Merville.

53 records · Page 3Linked to original sources
PubMed1995

Highly-expressed p100/p52 (NFKB2) sequesters other NF-kappa B-related proteins in the cytoplasm of human breast cancer cells.

Several observations have suggested that NF-kappa B transcription factors could be involved in carcinogenesis. To investigate the possibility that members of the NF-kappa B family participate in the molecular control of the transformed phenotype, we examined the expression of these proteins in human breast cancer cell lines as well as in primary tumors. Western Immunoblots demonstrated high expression of the p52 precursor p100 (NFKB2) in several breast cancer cell lines while human mammary epithelial cells express this protein only faintly. Eighteen primary breast tumors out of 24 displayed significant expression of the p100/p52 protein. In MDA-MB-435 cells, overexpressed p100 and p52 are predominantly cytoplasmic and coimmunoprecipitation experiments demonstrated that p100 sequesters the heterodimer p50/p65 in the cytoplasm. We demonstrate that most p65 protein is complexed with p100 in these cells while it is complexed predominantly with I kappa B-alpha in cell lines expressing less p100. Our data strengthen the hypothesis that NF-kappa B could be involved in carcinogenesis and suggest that the p100/p52 NF-kappa B subunit could play a role in the development of human breast cancers, possibly by sequestering other NF-kappa B-related proteins in the cytoplasm.

Adenocarcinoma↗
PubMed1995

Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Amino Acid Sequence↗
PubMed1995

Tamoxifen and its active metabolite inhibit growth of estrogen receptor-negative MDA-MB-435 cells.

Tamoxifen (TAM), the non-steroidal anti-estrogen most widely administered to breast cancer patients, acts, at least in part, by competing with estrogen receptors (ER). However, the existence of an alternative mechanism of action for this drug is supported by the clinical observations that: (a) 30% of patients with ER-negative cancer cells respond to TAM, and (b) 30% of patients with ER-positive cancer cells are not sensitive to this anti-estrogen. In this study, we observed that growth of the human ER-negative breast cancer cell line MDA-MB-435 was inhibited by TAM and 4-hydroxytamoxifen (4OH-TAM) in a concentration-dependent fashion. Both monoclonal enzymoimmunoassay and Dextran Charcoal Coated Scatchard radioimmunoassay analysis demonstrated that this MDA-MB-435 cell line does not express ER. The absence of ER in MDA-MB-435 cells was also demonstrated at the mRNA level by both northern blot hybridization and reverse transcription-polymerase chain reaction techniques. MDA-MB-435 cell proliferation was not affected by 17 beta-estradiol or by the pure anti-estrogen ICI 164384, further demonstrating that the observed effects of TAM and its active metabolite on the proliferation of MDA-MB-435 cells were due to an ER-independent mechanism, yet to be identified. MDA-MB-435 thus appears to be a promising original model for the study of the alternative ER-independent mechanisms of action of TAM.

Breast Neoplasms↗
PubMed1995

Varicella-zoster virus gene regulation.

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter.

Chromosome Mapping↗
PubMed1994

Expression of bone sialoprotein, a bone matrix protein, in human breast cancer.

Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. To date, the molecular mechanism that leads to the deposition of hydroxyapatite in the mammary tissue has not been elucidated. Bone sialoprotein (BSP) is a glycoprotein the expression of which coincides with the appearance of the first hydroxyapatite crystals during bone development. In this study, we report the observation that BSP, a bone matrix protein, is expressed in human mammary cancer cells. Using an immunoperoxidase technique, we studied the expression of BSP in 79 breast lesions, including 28 benign and 51 malignant specimens. Two polyclonal antibodies, one directed against intact human BSP and the other against a synthetic peptide of BSP (residues 277-294), were used and gave identical results. Normal mammary glands expressed undetectable or barely detectable amounts of BSP, and the majority of the benign lesions examined were generally unstained (0) or weakly stained (1+). Most of the breast carcinoma specimens (around 87%) showed a significant increase (P = 0.0001) in BSP expression. Breast carcinomas with microcalcifications had the highest immunoreactivity (2+ or 3+) to BSP antibodies. This is the first demonstration that BSP expression is significantly increased in breast cancer. Expression of BSP by breast cancer cells could play a major role in the deposition of microcalcifications and in the preferred bone homing of breast cancer cells.

Breast↗
PubMed1994

The NF-kappa B transcription factor and cancer: high expression of NF-kappa B- and I kappa B-related proteins in tumor cell lines.

NF-kappa B is a pleiotropic transcription factor which controls the expression of many genes and viruses. To date, there is good evidence, but no definitive proof, for its role in tumor formation and development of metastasis. To investigate the possibility that members of the NF-kappa B family could participate in the molecular control of the transformed and invasive phenotype, we examined the expression of these proteins in a variety of human tumor cell lines. The expression of p50, p65, p52 and I kappa B was quantified at the protein level using western immunoblot and mobility shift assay and at the RNA level by northern blot. We observed high expression of the NF-kappa B inhibitor I kappa B in the ovarian carcinoma cell line OVCAR-3 together with constitutive nuclear NF-kappa B activity. We also studied the colon carcinoma cell line HT-29 and its metastatic counterpart HTM-29 and we observed specific expression of the p52 NF-kappa B-related protein in the metastatic cells. Our data confirm that NF-kappa B could be involved in the genesis of a variety of cancers including solid tumors and provide us with interesting models to explore the exact role of these transcription factors in cancer.

Cell Nucleus↗
PubMed1994

[Tamoxifen in the treatment of breast cancer].

Tamoxifen is the most often prescribed non steroidal antioestrogenic agent in the world for breast cancer. Worldwide collaboration. has centralized the results, of different trials throughout the world on oral adjuvant therapy in the early stages of breast cancer. A significative regression of the tumour was observed in most cases. Moreover, recent epidemiological studies suggest that tamoxifen could prevent new contralateral primary tumours. The risk of the disease should thus be reduced by the prophylactic use of antioestrogens such as tamoxifen. Investigations using a variety of models have evaluated the effect of tamoxifen on tumour promotion and cell growth. Tamoxifen-induced growth inhibition is associated with major changes in biochemical events in cultured human breast cancer cells including cell proliferation or growth factor production. Growth inhibition of oestrogen-responsive human breast cancer cells is associated with an induced secretion of autoinhibitory polypeptides (TGF beta) and an antagonistic effect on the synthesis of proliferative proteins (TGF alpha,...). The first step in the mechanism of action of the drug is binding of tamoxifen to the oestrogen receptors. Development of resistance to tamoxifen treatment is a great problem in treatment of breast cancer patients and the mechanism of resistance will require further study: under the influence of the drug, tumours could become remodelled as selected subpopulations emerge resistant-tamoxifen. The fact that some breast cancers which are oestrogen receptor-negative respond to antioestrogen suggests that parallel but separate pathways for oestrogen and antioestrogen action may exist. This paper summarizes the results of the most recent studies concerning this promising drug.

Breast Neoplasms↗
PubMed1993

Stimulation of the 92-kD type IV collagenase promoter and enzyme expression in human melanoma cells.

The 92-kD type IV collagenase is a member of the metalloproteinase family which degrades type IV collagen, a major component of basement membrane and is involved in tumor invasion and metastasis. The promoter and adjacent regulatory sequences of the 92-kD type IV collagenase have been identified previously and three cis-acting elements homologous to the binding sites for AP-1, NF-KB and SP-1 proteins contributed to induction of the promoter activity by 12-O-tetradecanoylphorbol 13-acetate (TPA) and tumor necrosis factor (TNF-alpha) in HT1080 cells. To date, no direct correlation between promoter activity and expression of the 92-kD type IV collagenase has been reported in normal or cancer cells. In this study, the effects of the transcriptional stimulation of the 92-kD type IV collagenase gene on the expression of the enzyme in human A2058 melanoma cells was analyzed by zymography experiments. Quantitative immunoblots using a monoclonal antibody that recognized specifically and exclusively the 92-kD type IV collagenase, confirmed that the 92-kD gelatinase was 92-kD type IV collagenase. Stimulation of the promoter activity resulted in increased gelatinase activity in the culture medium of A2058 cells. A direct correlation between TPA- and TNF-alpha-mediated promoter stimulation of the 92-kD type IV collagenase gene and its expression was also demonstrated in the human fibrosarcoma HT1080 cells. Interleukin-1 alpha failed to induce 92-kD gene promoter activity and type IV collagenase expression in melanoma and fibrosarcoma cell lines. Our data demonstrated that TPA- and TNF-alpha-induced 92-kD type IV collagenase promoter stimulation leads to a proportional increase of enzyme expression and secretion and thus could contribute to the activation of the invasive phenotype.

Base Sequence↗
PubMed1986

Termini generated at the site of the DNA breakage mediated by photoexcited promazines.

Promazine derivatives are known to be able to photoinduce, in vitro, direct single-strand breaks into DNA (Decuyper et al., Biochem. Pharmac. 33, 4025-4031 (1984]. Using [32P]end labeled DNA fragments, it is demonstrated that this DNA breakage occurs almost regardless of the nucleotide sequence of the DNA. Using 3'-[32P]end or 5'-[32P]end labeled oligonucleotide and enzymatic digestion of the fragments generated, it is demonstrated that the termini generated at the site of the breakage are 5'-phosphate, 3'-phosphate and 3'-termini which are presumed to be 3'-phosphoglycolate. This is consistent with an attack of the sugar moeity of the sugar-phosphate backbone of the DNA by the reactives species generated upon near-u.v. irradiation of promazine derivatives.

DNA↗
PubMed1984

Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines.

Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were primed either with restriction fragments or synthetic primers and irradiated with near UV light in the presence of promazine derivatives. These DNAs were used as template for in vitro complementary chain synthesis by Escherichia coli DNA polymerase I large fragment. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis of the synthesis products and localized by comparison with a standard dideoxy sequencing pattern. More than 90% of the chain terminations were mapped exactly one nucleotide before a guanine residue. In addition, photoreaction was shown to occur more predominantly with guanine residues localized in single-stranded parts of the genome. The same guanine residues could also be damaged when the reaction was performed, in the dark, in the presence of the artificially generated promazine cation radicals. Using the BamHI-SmaI adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a covalent addition of the promazine, or of a cation radical photodegradation product, on the guanine moiety. Kinetics of chlorpromazine photoaddition on single-stranded and double-stranded DNAs were determined.

Acepromazine↗
PubMed1984

Induction of breaks in deoxyribonucleic acid by photoexcited promazine derivatives.

Near-u.v. photoexcited promazine and three of its derivatives are shown to induce single-strand breaks in phi X174-DNA replicative form. The mechanisms of this DNA breakage depend upon the various photochemical properties of the promazine derivatives. Chlorpromazine is shown to act predominantly via the photodechlorination reaction both in aerobic and anaerobic conditions. The three other promazine derivatives (promazine, trifluopromazine and methoxypromazine) display two mechanisms for DNA breakage. One of them occurs through the cation radical, which is formed during near-u.v. irradiation of promazine derivatives. The second mechanism is demonstrated to act via an hydroxyl radical-dependent pathway. Acepromazine is without photoactivated action. EPR-spin-trapping studies of irradiated mixtures, containing the drugs and 5,5-dimethyl-1-pyrroline-N-oxide (as spin trap), suggest the production of superoxide radical by photoexcited promazines. When DNA is present in the irradiation mixture, this superoxide radical is converted into hydroxyl radical probably via a Haber Weiss-type reaction, catalysed by DNA-iron complexes.

DNA↗
PubMed1984

In vitro cross-linking of bovine lens proteins photosensitized by promazines.

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O2, as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [ TFPZ ], methoxypromazine [ MTPZ ], and acepromazine [ ACPZ ] ), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo.

Acepromazine↗
PubMed1983

Lack of singlet oxygen formation by photoexcited promazine derivatives in aqueous and ethanolic solutions.

The EPR detection of nitroxide formation and the observation by thin layer chromatography of the specific 1O2 oxidation product of cholesterol, have been used to appreciate singlet oxygen production by promazine and four of its derivatives during irradiation with near-UV light of ethanolic and aqueous solutions. Within the range of sensitivity of the methods, no 1O2 had been detected.

Acepromazine↗
PubMed1983

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage øX174.

Inactivation of øX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a øXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN3 on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N2 atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.

Azides↗
PubMed1983

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins.

Irradiation in the presence of O2, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min-1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ). A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN3; (iii) NaN3 similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers. In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.

Animals↗