Clonal derivatives of a human B-lymphoblastoid cell line producing prolactin—A cytogenetic characterization

The IM-9-P cell line is a variant of the human B-lymphoblastoid cell line IM-9 which ectopically secretes prolactin (hPRL). The heterogeneous line IM-9-P and three sublines of clonal origin, two of them positive and one negative for PRL gene expression, were subjected to cytogenetic analysis and compared with the reference line IM-9 which showed a normal female diploid karyotype. G-banding revealed several rearrangements in the chromosomes. Nine altered chromosomes including one stable marker chromosome were common to all analysed karyotypes of IM-9-P cells and their clones. A second marker chromosome mar2 occurred only in the karyotypes of the hPRL producing clones, but not in the non-producing clone. None of the visible alterations involve chromosome 6 which carries the PRL gene in humans.     

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.     

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Alterations in c-abl gene methylation in cells transformed by phagocyte-generated oxidants

DNA from 10T1/2 cells transformed by activated neutrophils was analyzed for restriction length polymorphisms (RFLPs) in cellular homologues of retroviral oncogenes, and consistent RFLPs were found in MspI sites of the c-abl gene of all PMN-transformed cell lines. MspI digests probed with c-myc, v-Ki-ras, v-Ha-ras or v-mos showed no RFLPs, and none were observed in EcoRI, PstI, HindIII, BamHI, SmaI, Sau3a, MboI, HhaI, or TaqI digests probed with v-abl. Analysis of HpaII digests supports the conclusion that c-abl RFLPs result from differential methylation of the CCGG HpaII/MspI recognition sequence. MspI RFLPs in the c-abl gene may provide markers for oxidant-related genetic injury.     

Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation

Four clonal cell lines subcloned from a clonal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of alpha 1(I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases MspI and HpaII. It was found that the loss in collagen type I expression correlated positively with the degree of methylation of alpha 1(I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen gene regulation.