scholarly journals DNA methylation inhibits transcription of procollagen α2(I) promoters

1992 ◽  
Vol 283 (3) ◽  
pp. 699-703 ◽  
Author(s):  
D K Guenette ◽  
J D Ritzenthaler ◽  
J Foley ◽  
J D Jackson ◽  
B D Smith
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Transcriptional Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Parent Cell ◽  
Transcriptional Inhibition ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Hpaii Site ◽  
Exon Region

Our previous studies have demonstrated that a 2-[N-(acetoxyacetyl)amino]fluorene-transformed rat epithelial-like cell line, W8, contains a transcriptionally inactive alpha 2(I) gene with a hypermethylated promoter/first-exon region. We have cloned the rat promoter/first-exon region (-211 to +207) from W8 cells and their parent cell line, K16, which expresses alpha 2(I) collagen. There were no sequence differences between the clones from the two cell lines, indicating that a mutation was not responsible for transcriptional inhibition. The alpha 2(I) rat promoters were cloned upstream of the chloramphenicol acetyltransferase gene. Both constructs were equally active in both cell lines, suggesting that trans-activating factors for alpha 2(I) transcription are present in W8 cells. Finally, methylation of plasmids at all CpG sites with SssI methylase completely inhibited transcription using alpha 2(I) promoters, but methylation did not inhibit simian-virus-40 promoter-driven transcription. Certain methylation sites partially inhibit promoter activity. An HhaI methylation site inhibited transcriptional activity of the alpha 2(I) promoter 8-fold, whereas methylation at the HpaII site in the rat alpha 2(I) promoter did not decrease transcriptional activity. This provides further evidence that methylation at specific sites in the collagen alpha 2(I) promoter is responsible for the inactivation of transcription in W8 cells.

scholarly journals Transcriptional promiscuity of the human alpha-globin gene.

1989 ◽  
Vol 9 (1) ◽  
pp. 241-251 ◽  
Author(s):  
E Whitelaw ◽  
P Hogben ◽  
O Hanscombe ◽  
N J Proudfoot
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Multiple Copies ◽  
Positive Effect

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

scholarly journals Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

1986 ◽  
Vol 6 (6) ◽  
pp. 1974-1982 ◽  
Author(s):  
I Garcia ◽  
B Sordat ◽  
E Rauccio-Farinon ◽  
M Dunand ◽  
J P Kraehenbuhl ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Doubling Time ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Oncogenic Potential ◽  
Transformed Cell Line ◽  
Epithelial Cell Lines ◽  
Transforming Genes

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

scholarly journals Neoplastic transformation of rat 3Y1 cells by a transcriptionally activated human c-myc gene and stabilization of p53 cellular tumor antigen in the transformed cells.

1986 ◽  
Vol 6 (12) ◽  
pp. 4379-4386 ◽  
Author(s):  
K Shiroki ◽  
K Segawa ◽  
Y Koita ◽  
M Shibuya
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Transformed Cells ◽  
Agar Culture ◽  
Myc Gene ◽  
Tumorigenic Cell Line

Transformed foci were obtained in rat 3Y1 fibroblasts cotransfected with pRmyc 27 (transcriptionally activated c-myc) and pSV2neo DNA. RmycY cell lines (1 to 7) were established from these foci. RmycY cells were small and round and contained enlarged nucleoli in the nucleus. The myc gene was expressed in these cell lines at a much higher level than in 3Y1 cells and at a level similar to that in HL-60 cells. These cell lines formed colonies in soft-agar culture and tumors in syngeneic rats transplanted with RmycY cells. Expression of the gene and colony formation in soft-agar culture were analyzed in subclones from RmycY cell line 1. A correlation between myc gene expression and the ability to form colonies in soft-agar culture was observed in these cells. Antibody against p53 cellular tumor antigen was detected in some sera from tumor-bearing rats. p53 cellular tumor antigen stabilized and accumulated in RmycY cells to the same extent as in simian virus 40-transformed cells. The results suggest that elevated c-myc expression and an increased amount of p53 cause 3Y1 cells to become a more tumorigenic cell line.

scholarly journals ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS

1973 ◽  
Vol 58 (1) ◽  
pp. 11-26 ◽  
Author(s):  
Wolfgang Klietmann ◽  
Nobuhiro Sato ◽  
Margit M. K. Nass
Keyword(s):  
Mitochondrial Dna ◽  
Cell Line ◽  
Cell Lines ◽  
Ethidium Bromide ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Ultrastructural Changes ◽  
Parental Cell Line ◽  
Mammalian Cell Lines

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a3 show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.

scholarly journals Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes

1986 ◽  
Vol 6 (6) ◽  
pp. 1974-1982
Author(s):  
I Garcia ◽  
B Sordat ◽  
E Rauccio-Farinon ◽  
M Dunand ◽  
J P Kraehenbuhl ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Doubling Time ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Oncogenic Potential ◽  
Transformed Cell Line ◽  
Epithelial Cell Lines ◽  
Transforming Genes

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Neoplastic transformation of rat 3Y1 cells by a transcriptionally activated human c-myc gene and stabilization of p53 cellular tumor antigen in the transformed cells

1986 ◽  
Vol 6 (12) ◽  
pp. 4379-4386
Author(s):  
K Shiroki ◽  
K Segawa ◽  
Y Koita ◽  
M Shibuya
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Transformed Cells ◽  
Agar Culture ◽  
Myc Gene ◽  
Tumorigenic Cell Line

Transformed foci were obtained in rat 3Y1 fibroblasts cotransfected with pRmyc 27 (transcriptionally activated c-myc) and pSV2neo DNA. RmycY cell lines (1 to 7) were established from these foci. RmycY cells were small and round and contained enlarged nucleoli in the nucleus. The myc gene was expressed in these cell lines at a much higher level than in 3Y1 cells and at a level similar to that in HL-60 cells. These cell lines formed colonies in soft-agar culture and tumors in syngeneic rats transplanted with RmycY cells. Expression of the gene and colony formation in soft-agar culture were analyzed in subclones from RmycY cell line 1. A correlation between myc gene expression and the ability to form colonies in soft-agar culture was observed in these cells. Antibody against p53 cellular tumor antigen was detected in some sera from tumor-bearing rats. p53 cellular tumor antigen stabilized and accumulated in RmycY cells to the same extent as in simian virus 40-transformed cells. The results suggest that elevated c-myc expression and an increased amount of p53 cause 3Y1 cells to become a more tumorigenic cell line.

Transcriptional promiscuity of the human alpha-globin gene

1989 ◽  
Vol 9 (1) ◽  
pp. 241-251
Author(s):  
E Whitelaw ◽  
P Hogben ◽  
O Hanscombe ◽  
N J Proudfoot
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Multiple Copies ◽  
Positive Effect

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

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