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Biomedical subjects

S Mai

Publications and source records attributed to S Mai.

At least 37 records · Page 2Linked to original sources
PubMed1997

c-Myc dependent initiation of genomic instability during neoplastic transformation.

The dihydrofolate reductase (DHFR) gene is a target of c-Myc in genomic instability. The induced overexpression of c-Myc in cell lines is followed by the amplification and rearrangement of the DHFR gene. Furthermore, the constitutive upregulation of c-Myc protein coincides with genomic instability of the DHFR gene in lymphoid, non-lymphoid and in tumor lines. The amplification of the DHFR gene is locus-specific and independent of species origins. We have now addressed the question whether inducible deregulation of c-Myc is followed by DHFR gene amplification in vivo. We show that the DHFR gene is a target of c-Myc-dependent neoplasia in vivo and propose a role for genomic instability during the initiation of neoplastic transformation.

Animals↗
PubMed1997

Genomic instability and apoptosis are frequent in p53 deficient young mice.

The loss of p53 tumor suppressor functions results in genetic instability, characteristically associated with changes in chromosome ploidy and gene amplification. In vivo, we find that cells from various organs of 4 to 6-week old p53-nullizygous (p53-/-) mice display aneuploidy and frequent gene amplification as well as evidence for apoptosis. Regardless of tissue types, many p53-/- cells contain multiple centrosomes and abnormally formed mitotic spindles. Thus, chromosome instability in vivo may be associated with abnormal centrosome amplification. Moreover, we observed a significant increase in the number of cells overexpressing c-Myc in p53-/- mice. Consistent with previous studies showing that c-Myc overexpression is associated with gene amplification in vitro, many of the p53-/- cells exhibited, in the same cell, c-Myc overexpression and amplified c-myc, dihydrofolate reductase (DHFR), and carbamoyl-phosphate synthetase-aspartate transcarbamoyl-dihydroorotase (CAD) genes. Furthermore, apoptosis was frequently observed in cells isolated from p53-/- mice. The apoptotic cells contained abnormally amplified centrosomes, displayed aneuploidy, high levels of c-Myc expression, as well as gene amplification. These results indicate that a high number of aberrant cells is eliminated by p53-independent pathways in vivo.

Amino Acid Sequence↗
PubMed1997

C-myc overexpression facilitates radiation-induced DHFR gene amplification.

To investigate the role of myc-overexpression on radiation-induced amplification of the dihydrofolate reductase gene (DHFR) we compared diploid Chinese hamster ovary cells (CHO-9) to cells of the same line that had been stably transfected with a dexamethasone-inducible c-myc cDNA. The application of flow-cytofluorometry and fluorescent in situ hybridization (FISH) allowed the evaluation of an increase in DHFR gene copy number following radiation treatment without the use of a preceding selection procedure. We show that DHFR gene amplification may occur independently of p53 status in cells overexpressing c-myc.

Animals↗
PubMed1997

[What is the current age and sex-related cadmium burden of the human kidney cortex?].

55 persons of either sex in the age bracket between 0.02 and 87 years had to be examined by experts in forensic medicine. In these examinations, slices of kidney cortex were removed and prepared for cadmium(Cd)-determination by atomic absorption spectrophotometry. The samples were wet ashed by a special and quick method developed in our laboratory, using concentrated (65%) HNO3 for a short period of 1 hour at 90 degrees C. The main results of the present study were that Cd accumulation in the kidney cortex was delayed in the age group between 0.02 and 40 years. The highest Cd concentration of 27 micrograms/g wet weight was found in the age group between 50-59 years. In persons of higher age, above 60 years, the Cd concentrations were generally lower than in persons of the age group between 50-59 years; however, this decline does not seem to be significant. Summing up, we can say that the delayed increase in Cd concentrations in the kidney cortex of persons in the age group between 0.02 and 40 years might be regarded as the first positive result of the restrictive use of Cd materials, resulting in food intake with lower Cd contamination. The present study should be repeated after about 5 years to confirm whether this hopeful assumption is true.

Adolescent↗
PubMed1996

c-Myc overexpression associated DHFR gene amplification in hamster, rat, mouse and human cell lines.

We have analysed relative DHFR gene copy numbers in nine cell lines of various cell type and species origins. The cells studied expressed either low, low and inducible or constitutively elevated levels of c-Myc protein. DHFR gene amplification was observed only when c-Myc protein levels were upregulated. The amplification of the DHFR gene was transient in inducible cell lines. Cell lines exhibiting constitutively deregulated c-Myc protein levels, however, showed both DHFR gene amplification and ongoing rearrangements of the DHFR locus. In contrast, the relative gene copy numbers of ribonucleotide reductase R1 subunit, ornithine decarboxylase, syndecan 2, glyceraldehyde-3-phosphate-dehydrogenase, and cyclin C remained unaffected irrespective of c-Myc protein levels, suggesting a locus-specific genomic instability of the DHFR gene in cells with deregulated c-Myc protein levels. Overall, the results of the present study support the notion that DHFR gene amplification as a consequence of c-Myc deregulation may occur in a variety of cell lines irrespective of their cell type and species origins.

Animals↗
PubMed1996

Genomic instability in MycER-activated Rat1A-MycER cells.

The deregulated expression of c-Myc protein is associated with the non-random locus-specific amplification of the dihydrofolate reductase (DHFR) gene. This study was performed to determine whether additional chromosomal aberrations occur when c-Myc protein levels are up-regulated for prolonged periods. To this end, we have used Rat1A-MycER cells, which allow the experimental regulation of Myc protein levels. We examined the genomic stability of Rat1A-MycER cells cultivated in either the absence or the presence of estrogen, which reportedly activates the chimeric MycER protein in these cells. Following prolonged periods of MycER activation, Rat1A-Mycer cells exhibited irreversible chromosomal aberrations. The aberrations included numerical changes, chromosome breakage, the formation of circular chromosomal structures, chromosome fusions, and extrachromosomal elements.

Animals↗
PubMed1995

Enhanced p53 activity and accumulation in response to DNA damage upon DNA transfection.

In response to DNA damage the wild-type tumor suppressor protein p53 accumulates in the nucleus of rodent and primate cells. To investigate the minimal requirement for this reaction the cellular DNA was restricted by two alternative ways: (i) by calicheamicin gamma 1, an enediyne, which causes direct, sequence-specific DNA damage, as shown by fluorimetric analysis of DNA unwinding and by poly(ADP-ribose) polymerase activation. The dose-dependent DNA damage correlated with the nuclear p53 accumulation. In addition, restriction was generated (ii) by the intracellular introduction of the restriction enzyme PvuII, which generates blunt-ended DNA breaks, applying a mild hypotonic shock (pellet method). Previous transfection of linear or circular, single- or ds, DNA, followed by mitomycin C-treatment, lead to a dramatic increase in nuclear p53 accumulation and p53 activity according to electrophoretic mobility shift analysis. The nature of transfected DNA was irrelevant for enhanced accumulation. The data suggest, that the cellular p53 response to DNA damage is sensitized by uptake of exogenous DNA.

Aminoglycosides↗
PubMed1995

The c-myc protein represses the lambda 5 and TdT initiators.

The lambda 5 promoter initiates transcription at multiple sites and confers expression in all cell types. Two lambda 5 promoter-derived oligonucleotides (Inr lambda 5:1 and Inr lambda 5:2), each with a transcription start site, could promote transcription in transient transfection assays. In contrast, a third oligonucleotide (+90 lambda 5), without a transcription initiation site, was inactive. The Inr lambda 5:1 and Inr lambda 5:2 oligonucleotides formed a major DNA-protein complex B' in gel retardation analyses; no protein-DNA complexes were observed with the inactive +90 lambda 5 oligonucleotide. The B' complexes of Inr lambda 5:1 and Inr lambda 5:2 each contained c-myc and myn (murine homologue of Max) proteins. The c-myc and myn proteins were also found to bind the TdT initiator (InrTdT). Using mutated oligonucleotides, we found that the c-myc/myn proteins bound to the transcription initiation site of both Inr lambda 5:1 and InrTdT, however, these mutated oligonucleotides were inactive in transfection assays. This suggested that, in this system, transcription depended both on a transcription initiation site and appropriate flanking sequences. The significance of c-myc binding to the respective initiator was analysed by overexpressing c-myc in co-transfection assays. Under these conditions the transcriptional activity of both the lambda 5 and the TdT initiator was repressed.

Amino Acid Sequence↗
PubMed1995

Amplified extrachromosomal elements containing c-Myc and Pvt 1 in a mouse plasmacytoma.

After adaptation of a mouse plasma cell tumor, MOPC265, to culture, we have found several unique chromosomal alterations in addition to the T(12;15) translocation and trisomy 11 frequently observed in plasmacytomas. Among these alterations is a specific coamplification of the c-Myc and Pvt 1 gene loci from mouse chromosome 15. Further analysis by fluorescence in situ hybridization demonstrates that the amplicons of c-Myc and Pvt 1 exist as extrachromosomal elements as well as within intact chromosomes. Most importantly, the presence of both Pvt 1 and c-Myc in these extrachromosomal elements indicates ongoing coselection for these loci in the propagation of MOPC265.

Animals↗
PubMed1994

Overexpression of c-myc precedes amplification of the gene encoding dihydrofolate reductase.

An experimentally inducible model system was generated in which Chinese hamster ovary cells (CHO-9) were stably transfected with an inducible c-myc cDNA. The induction of c-myc in these transfectants is followed by the enhanced binding of c-Myc/Max-containing protein complexes to 5'flanking E-box sequences of the gene encoding dihydrofolate reductase (DHFR). Moreover, DHFR is transiently amplified. The inappropriate overproduction of the oncoprotein, therefore, seems to plays a role in induced DHFR amplification.

Animals↗
PubMed1994

c-Myc binds to 5' flanking sequence motifs of the dihydrofolate reductase gene in cellular extracts: role in proliferation.

The dihydrofolate reductase is a key enzyme of the folate metabolism which supplies the cell with dTTPs for DNA synthesis. Using cellular extracts, we demonstrate the formation of c-Myc/Max heterodimers at the dihydrofolate reductase (DHFR) 5' flanking CANNTG (E-box) motifs. The presence of these complexes correlates with c-Myc levels and active cellular proliferation.

Amino Acid Sequence↗
PubMed1994

Fos and Jun form cell specific protein complexes at the neuropeptide tyrosine promoter.

In this study we have investigated DNA-protein interactions at an AP1-like motif of the neuropeptide tyrosine (NPY) promoter during in vitro differentiation of human neuroblastoma cells SH-SY5Y to mature nonproliferative sympathetic neuron-like cells. These neuroblast-like cells originate from the parental cell line SK-N-SH from which two phenotypically distinct major cell types have been subcloned: the neuroblast-like SH-SY5Y cells and the epithelial-like SH-EP cells. SH-SY5Y cells can be induced to differentiate towards mature noradrenergic ganglion-like cells by the protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate). Interestingly, the effects of TPA are mimicked by the protein kinase inhibitor, staurosporine, which induces the expression of TPA target genes such as the neuronal differentiation-associated gene NPY in SH-SY5Y cells. Following activation of PKC, the effects of TPA are known to act through the transcription factor AP-1. To study transcriptional regulation during sympathetic differentiation of human neuroblastoma cells by TPA as well as by staurosporine, we focussed on protein complexes at an evolutionarily conserved AP-1 like motif located at nucleotide positions -70 to -65 within the 5'-flanking region of the NPY gene. We show that both c-Jun and c-Fos are part of the protein complexes that bind to this sequence in SH-SY5Y cells. Both staurosporine and TPA enhanced and modulated the binding of these DNA-protein complexes concomitant with the NPY mRNA expression. On the other hand, the absence of these complexes in the SH-EP subclone was associated with the absence of NPY mRNA expression and a lack of differentiation-associated morphological changes. The data suggest that Fos and Jun heterodimers are part of the protein complexes that bind to the AP-1 regulatory element of the NPY promoter in the neuroblast-like SH-SY5Y cells. These protein complexes appear to contribute to the cell specific expression of the NPY gene and seem to be required during differentiation of SH-SY5Y human neuroblastoma cells further along the sympathetic neuronal lineage induced by either TPA or staurosporine.

Alkaloids↗
PubMed1991

[A choledochal diverticulum. A case report].

Due to the rarity of choledochal cystic dilatation, the authors report a clinical case of common bile duct diverticulum. This diverticulum was histologically composed of gastric ectopic mucosa (pyloric type) and it clinically caused chronic abdominal pain (in a young woman). The authors analyse the literature of the above pathology.

Adult↗
PubMed1990

Radiation-induced activation of transcription factors in mammalian cells.

In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation-induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s).

Animals↗
PubMed1989

UV-induced early-domain binding factor as the limiting component of simian virus 40 DNA amplification in rodent cells.

UV radiation and other carcinogenic agents induce an increase in DNA-binding activity to the early domain of the simian virus 40 (SV40) minimal origin in both SV40-permissive and SV40-nonpermissive cells. The increase is due to posttranslational modification of a preexisting protein, since it occurs in the presence of cycloheximide or anisomycin. Binding of this factor is an absolute requirement for the UV-induced SV40 DNA amplification in Co631 cells in vivo. A synthetic double-stranded oligonucleotide covering the early domain sequence totally blocked the UV-induced amplification in competition experiments. Point mutants of the sequence and unrelated oligonucleotides which could not bind the factor also did not block SV40 amplification. Inhibitors of protein synthesis caused an immediate increase of both early-domain factor activity (perhaps by prolonging mRNA half-life for the factor or for a modifying enzyme) and DNA amplification. The effects of UV and cycloheximide on SV40 amplification were superaddition.

Animals↗
PubMed1984

Cell attachment and spreading on extracellular matrix-coated beads.

Parietal yolk sac cells M1536 -B3 grown on cytodex 2 beads deposited an extracellular matrix on the surface of the beads. Cell-free matrix-coated beads were isolated by treatment of the cell monolayer with cytochalasin B (CB) at a concentration of 10 micrograms/ml of phosphate-buffered saline (PBS). The matrix when analysed by electrophoresis on polyacrylamide gels (PAGE) revealed that the major components were laminin and entactin . The matrix-coated beads were used to study the attachment, spreading, and growth of African Green monkey BSC-40, human mammary MCF-7, mouse fibroblast L929, rat liver clone 9, and rat hepatoma H-4-II-E cells in defined serum-free growth medium. The different cell lines exhibited varying responses to matrix-coated vs uncoated beads with respect to rate of attachment, spreading, and growth. One of the most consistent responses observed was the enhancement of cell spreading on matrix-coated beads. The results suggested that the matrix-coated beads will provide a readily available and valuable tool for studies on cell surface-extracellular matrix interactions and the physiological consequences of those interactions.

Animals↗