ABSTRACT
Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.
Subject(s)
Breast/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Epithelial Cells/metabolism , Laminin/biosynthesis , Transcription Factors/metabolism , Transcription Factors/physiology , Binding Sites , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , Down-Regulation , Gene Expression Regulation , Genes, Reporter , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors , Models, Genetic , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Time Factors , Transcription Factor AP-1/metabolism , Transcription, Genetic , Transfection , Tumor Cells, CulturedABSTRACT
We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes N-alkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2-24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased MPG protein expression in the tumor cells that correlated with elevated glycosylase activity. Since MPG overexpression has been shown to be paradoxically associated with increased susceptibility to DNA damage, up-regulation of this gene may suggest a functional role in breast carcinogenesis.
Subject(s)
Breast Neoplasms/enzymology , DNA Glycosylases , DNA Repair , N-Glycosyl Hydrolases/biosynthesis , Adenine/analogs & derivatives , Adenine/metabolism , Amino Acid Sequence , Breast/metabolism , Breast Neoplasms/genetics , Cells, Cultured , Humans , Molecular Sequence Data , N-Glycosyl Hydrolases/metabolism , RNA, Messenger/metabolism , Tumor Cells, CulturedABSTRACT
8-OH-deoxyguanosine can diminish the ability of the restriction endonucleases Hpa II and Msp I to cleave DNA. The exact position of the adduct within the recognition site appears to determine the extent of the effect.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA/metabolism , Deoxyguanosine/metabolism , Deoxyribonuclease HpaII/metabolism , Base Sequence , Free Radicals/metabolism , Humans , Molecular Sequence Data , Oligonucleotides , Reactive Oxygen SpeciesABSTRACT
8-Hydroxyl-2'-deoxyguanosine (also referred to as 8-hydroxyguanine [8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA via reactive oxygen species, affects the in vitro methylation of nearby cytosine moieties by the human DNA methyltransferase. The exact position of 8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of the methyltransferase to methylate a target cytosine when the 8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the same strand. On the other hand 8-OH-deoxyguanosine does not diminish the ability of the enzyme to respond to a methyl director (5-methylcytosine) when the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3' from the methyl director. Differences in methylation rates as great as 13-fold have been detected using various 8-OH-deoxyguanosine-containing oligonucleotides as substrates in methylation assays. Our findings suggest that oxidative damage of parental strand guanines would permit normal copying of methylation patterns through maintenance methylation, while oxidative damage of guanines in the nascent strand DNA would inhibit such methylation.
Subject(s)
DNA Adducts , DNA Modification Methylases/metabolism , Guanine/analogs & derivatives , Oligodeoxyribonucleotides , Base Sequence , Binding Sites , DNA-Cytosine Methylases/metabolism , Female , Guanine/pharmacology , Humans , Methylation , Molecular Sequence Data , Placenta/enzymology , Pregnancy , Reactive Oxygen SpeciesABSTRACT
Methylation of cytosines in DNA is important for the regulation of expression of many genes. During carcinogenesis, normal patterns of gene methylation can be altered. Oxygen radical injury, shown to damage DNA in a variety of ways associated with cancer development and other conditions, has been suggested to affect DNA methylation, but a mechanism has not been demonstrated. Using oligonucleotides containing the common oxygen radical adduct 8-hydroxyguanine to replace guanine, we found that the enzymatic methylation of adjacent cytosines is profoundly altered. Furthermore, there is a high degree of positional specificity with respect to this effect. Thus, free radical injury may explain some of the altered methylation observed during carcinogenesis.
