RESUMO
The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.
Assuntos
Mama/metabolismo , Senescência Celular/fisiologia , Aberrações Cromossômicas , Células Epiteliais/metabolismo , Divisão Celular , Linhagem Celular , Transformação Celular Neoplásica , Células Cultivadas , Metilação de DNA , Feminino , Fibroblastos/metabolismo , Humanos , Proteínas Oncogênicas Virais/metabolismo , Regiões Promotoras GenéticasRESUMO
Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.
Assuntos
Mama/citologia , Senescência Celular , Adolescente , Adulto , Mama/metabolismo , Mama/ultraestrutura , Divisão Celular , Transformação Celular Neoplásica , Células Cultivadas , Aberrações Cromossômicas , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Feminino , Fibroblastos/citologia , Humanos , Cariotipagem , Telômero , Proteína Supressora de Tumor p53/metabolismoRESUMO
Intrachromosomal homologous recombination has been revealed in the DNA from transgenic mice of three pedigrees. The recombination DNA of aminoglycosid phosphoribosiltransferase (neo) gene was observed in liver, kidney, spleen, heart, skeletal muscle, germinal glands and tail. It is concluded that the constructed model can be used for studying recombinations in cells of various organs and tissues both in the course of embryogenesis and during their malignant transformation and tumor progression.
Assuntos
Cromossomos/genética , DNA/genética , Regulação Enzimológica da Expressão Gênica/genética , Pentosiltransferases/genética , Recombinação Genética/genética , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
A study was made of the influence of sodium butyrate and luminol on the rate of homologous recombination of aminoglycosidephosphotransferase gene copies, which contain non-overlapping deletions and are integrated in chromosomes of the Chinese hamster cells of line A238. It is shown that treatment of cells of transformed clones with sodium butirate, which is known to stimulate DNA reparation and to loosen chromatin structure, inhibits intrachromosomal homologous recombination. Dexametazone, which is also capable of stimulating DNA reparation, displayed similar effect. In contrast, the treatment of cells with luminol, inhibiting DNA reparation and condensing chromatin structure, increased the efficiency of intrachromosomal homologous DNA recombination.
Assuntos
Cromatina/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , DNA/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacos , Homologia de Sequência do Ácido Nucleico , Animais , Butiratos/farmacologia , Ácido Butírico , Linhagem Celular , Cromatina/genética , Cromossomos/genética , Células Clonais , Cricetinae , Cricetulus , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , DNA/genética , Dexametasona/farmacologia , Resistência a Medicamentos/genética , Gentamicinas/antagonistas & inibidores , Canamicina Quinase , Luminol/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Recombinação Genética/genéticaRESUMO
For determination of the extrachromosomal homologous DNA recombination efficiency, somatic cells of various lines have been transformed with plasmid DNAs which contain copies of neo-gene with non-overlapping deletions. Reconstruction of the neo-gene functional activity, which imparts a geneticin-resistant phenotype to cells, indicates that recombination has occurred. If dP1 and dR copies of the neo-gene are used, a single (reciprocal) exchange is necessary for reconstruction of the neo-gene by homologous DNA recombination, but a double exchange (gene conversion) is needed in the case of dP1 and dS copies. It is shown that in human cells of line HeLa and in mouse cells of line LMtk-, in contrast to the Chinese hamster cells of line A238, the frequency of double exchanges is comparable to that of the single DNA exchanges which is an evidence of participation in DNA recombination of gene conversion in addition to a single exchange mechanism. The treatment of cells with sodium butyrate and luminol exerts different influences on the rate of the single DNA exchanges and on that of gene conversion (double exchanges) in cells of lines LMtk- and HeLa, respectively. Essential distinctions in correlation of the single DNA exchange frequency and the gene conversion frequency in cells of the studied lines, and the possibility to distinguish between these mechanisms of recombination, under the treatment by sodium butyrate and luminol, may suggest the existence of two mechanisms of homologous DNA recombination in cultured animal cells, which function independently of one another, to a considerable extent.
Assuntos
Butiratos/farmacologia , DNA/efeitos dos fármacos , Herança Extracromossômica/efeitos dos fármacos , Conversão Gênica/efeitos dos fármacos , Luminol/farmacologia , Recombinação Genética/efeitos dos fármacos , Animais , Ácido Butírico , Linhagem Celular , Células Cultivadas , Cricetinae , DNA/genética , Humanos , Camundongos , Plasmídeos/genética , Mapeamento por Restrição , Transformação Genética/efeitos dos fármacosRESUMO
The frequencies of homologous extrachromosomal recombination, following plasmid transfection into cell lines differing in their tumour potential were measured. A 10-fold increase in extrachromosomal plasmid recombination has been reported in tumour cells (Ra-2, Jf-1) relative to immortal (Rat-2) and normal (REF). The differences between the normal and tumour cells are preserved in the in vitro recombination experiments (the recombination in the cell nuclei extracts). However, the recombination activity of nuclear extracts from the immortality transformed cells (Rat-2) is similar to that of nuclear extracts from the tumour cells.
Assuntos
DNA de Neoplasias/genética , Herança Extracromossômica/genética , Plasmídeos/genética , Recombinação Genética/genética , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , Escherichia coli/genética , Deleção de Genes , Genes Bacterianos/genética , Variação Genética/genética , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Transfecção/genética , Transformação Bacteriana/genética , Células Tumorais CultivadasRESUMO
A plasmid DNA family containing deletion variants of Neo-gene has been constructed for investigation of somatic cell homologous recombination. Experimental data are presented provided by studies of extrachromosomal recombination, recombination following plasmid transfection into somatic cells, and recombination in the nuclear extracts. The opportunities of application of the plasmid constructions in the different pattern homologous recombination experiments performed on mammalian cells have been discussed.
