Genetic suppression of tumor formation.
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Animals↗
Biomedical subjects
R Sager
Publications and source records attributed to R Sager.
Gene amplification: an example of accelerated evolution in tumorigenic cells.
During selection for methotrexate resistance, tumorigenic CHEF/16 cells and derivatives from CHEF/16 tumors underwent amplification of the dihydrofolate reductase gene (DHFR) at accelerated rates compared with closely related nontumorigenic CHEF/18 cells. "Dot blot" analysis showed that the CHEF/16 cells contained many more copies of the DHFR gene than did the CHEF/18 cells, when assayed at similar elevated levels of methotrexate resistance. Chromosome analysis of cell samples taken at several time points during amplification revealed large differences between the nontumorigenic CHEF/18 cells and the two tumorigenic cell lines. The tumorigenic cells developed few chromosome rearrangements over a 4-log increase in methotrexate resistance, other than increased length of a single chromosome, which was shown by in situ hybridization to contain most or all of the amplified DHFR gene copies. In contrast, the CHEF/18 cells underwent complex, progressive changes in almost every chromosome, and in situ hybridization suggested a dispersed pattern of gene amplification. The data support the hypothesis that unregulated amplification is a pathological process, occurring readily in neoplastic but rarely in normal cells, that together with other chromosomal disturbances contributes to the rapid evolution and progression of cancer.
Animals↗
Suppression of tumorigenicity in hybrids of normal and oncogene-transformed CHEF cells.
Somatic cell hybridization experiments were carried out to determine whether normal cells have the ability to suppress the transforming effects of a defined oncogene. A nontransformed Chinese hamster embryo fibroblast cell line (CHEF/18-dm2) was used as the normal parent, and a CHEF/18 transfectant carrying the human mutant c-Ha-ras (EJ) oncogene was used as the tumorigenic parent. Selected hybrids (L318 cell lines) were assayed for the presence of EJ DNA, for the p21 product of the c-Ha-ras gene, and for various indices of cell transformation. These hybrids exhibited a fibroblastic morphology similar to the normal parent, although they contained the EJ gene and expressed its p21 protein product at levels comparable with the transformed parent. They had a reduced capacity for anchorage-independent growth (plating efficiency in methylcellulose of less than 0.3-13%, as compared with greater than 90% for the transformed parent) and decreased tumor-forming ability in athymic mice. These findings show that normal CHEF/18 cells contain suppressor genes capable of inhibiting expression of the transformed phenotype, and tumor-forming ability, in the presence of an activated EJ oncogene.
Animals↗
Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.
The Chinese hamster embryo fibroblast cell line CHEF/18 is readily transfected by plasmid DNA. In the present transfection studies with CHEF/18 cells, focus formation induced by plasmids containing the mutant human c-Ha-ras gene EJ was compared with that of control plasmids without the EJ insert. The focus-forming activity of the transfected plasmid J132, a recombinant of the Harvey murine sarcoma virus LTR and the normal human c-Ha-ras1 in pBR322, also was assessed. Foci were recovered after transfection with either pSV2gpt or pSV2neo at about 10% the frequency obtained with the EJ-containing plasmids, and J132 gave a similar frequency, all well above background obtained with salmon sperm DNA. Whereas foci from transfection with EJ-containing plasmids contained the EJ DNA, no plasmid DNA was detected in either tumorigenic or tumor-derived cells from foci transfected with pSVgpt, pSVneo, or J132. Evidence that genomic changes were induced by plasmid transfection is based on finding chromosomal aberrations in all expanded foci and tumor-derived cells examined. The results suggest the occurrence of "hit-and-run" tumorigenesis induced by transient plasmid transfection.
Animals↗
Adipocyte conversion of CHEF cells in serum-free medium.
When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serum-free medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, greater than 90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.
Adipose Tissue↗
Adipocyte differentiation in CHEF cells.
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Adipose Tissue↗
Control of maternal inheritance by DNA methylation in chlamydomonas.
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Chlamydomonas↗
Genetic analysis of tumorigenesis: XVI. Chromosome changes in azacytidine- and insulin-induced tumorigenesis.
Chromosome changes accompanying differentiation and tumorigenesis in azacytidine- (azaC) and insulin-induced preadipocytes of the Chinese hamster embryo fibroblast cell line CHEF/18 are described. Karyotype analysis of 47 clones, subclones, and tumor-derived cells has shown that trisomy for chromosome 3q (mar 1) is characteristic of azaC preadipocytes but not of insulin preadipocytes. AzaC preadipocytes were consistently tumorigenic as well as trisomic for chromosome 3q, whereas most insulin preadipocytes were nontumorigenic and diploid. Only the few insulin preadipocytes that were tumorigenic were also trisomic for chromosome 3q. Among the tumor-derived cell lines recovered from azaC preadipocytes injected into nude mice, four had no additional chromosome changes except trisomy for 3q, as detected by karyotype analysis. Thus trisomy for 3q may be a sufficient chromosome change to induce tumor-forming ability in these cells. The rearrangements of chromosome 3 seen in this and other work pinpoint the trisomic region between the centromere and 3q5.
Animals↗
Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes.
In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation.
Base Sequence↗
Characterization of DNA methyltransferase from bovine thymus cells.
A DNA methyltransferase was partially purified from bovine thymus heavy cells. The enzyme has Mr 130 000, and introduces methyl groups from S-adenosylmethionine into the 5 position of cytosines in DNA. Sequence specificity analysis revealed that about 60% of the total methylation occurred in the 5'd(C-G)3' doublet. Single-stranded and hemi-methylated DNAs were methylated at an elevated rate by the enzyme. The kinetic analysis showed that the reaction obeys a random sequential mechanism. These results suggest that the enzyme serves primarily as a maintenance DNA methyltransferase.
Animals↗
Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of Chlamydomonas.
In Chlamydomonas, the maternal inheritance of chloroplast genes correlates with the differential methylation of chloroplast DNA (chlDNA) in females (mt(+)) but not in males (mt(-)). Our previous studies have supported our methylation-restriction model in which the maternal transmission is accounted for by the differential methylation in gametes which protects female but not male chlDNA from degradation during zygote formation. In the mutant me-1 [Bolen, P. L., Grant, D. M., Swinton, D., Boynton, J. E. & Gillham, N. W. (1982) Cell 28, 335-343], chlDNA of vegetative cells of both mating types is heavily methylated even before gametogenesis; nonetheless, maternal inheritance occurs in mutants as in wild type. To investigate the mechanism of maternal inheritance in the me-1 mutant, we have compared restriction fragment patterns after agarose gel electrophoresis of chlDNAs from mutant vegetative cells and gametes with those from wild type, by using a set of 32 restriction enzymes of which 17 were methylation-sensitive in this system. We find that additional methylation occurs during gametogenesis in the mutant female (mt(+)) but not in the corresponding male (mt(-)). Thus, gamete-specific, mating-type-specific methylation occurs in the me-1 mutant as in the wild type, consistent with our methylation-restriction model. In the me-1 mutant, gametic methylation occurs on a background of vegetative cell methylation not present in wild-type cells and irrelevant to the regulation of chloroplast inheritance. Comparison of the me-1 mutation with the mat-1 mutation [Sager, R., Grabowy, C. & Sano, H. (1981) Cell 24, 41-47] provides evidence for the existence of two different chlDNA methylation control systems: mat-1, linked to the mating type locus and regulating the mating-type-specific methylation that correlates with maternal inheritance, and me-1, unlinked to the mating type locus and unrelated to the regulation of maternal inheritance.
Journal Article↗
Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.
5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subclones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumor-forming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I- and Hpa II-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.
Adipose Tissue↗
Lysis of Chinese hamster embryo fibroblast mutants by human natural cytotoxic (NK) cells.
The nontransformed, nontumorigenic CHEF/18 Chinese hamster embryo fibroblast line, as well as nontumorigenic CHEF/18 mutants that had become anchorage independent or acquired a reduced serum requirement for growth, and fully transformed, tumorigenic CHEF cell lines were analyzed for their sensitivity to killing in vitro by human natural killer (NK) cells. Nontumorigenic but transformed anchorage-independent and low-serum-requiring mutants remained insensitive to NK-mediated lysis like the parent CHEF/18 line. Only fully tumorigenic CHEF lines were found to be sensitive to NK-mediated lysis, although a few tumorigenic lines were resistant to NK lysis. These results indicate that NK sensitivity is not the result of any cellular changes associated with acquisition of an anchorage-independent or low-serum-requiring phenotype but is the result of some additional change(s) found only in fully tumorigenic CHEF cells. Our studies also show that, whatever the NK target structure is, it is evolutionarily conserved so that human NK cells are able to distinguish between Chinese hamster tumorigenic and nontumorigenic cells.
Animals↗
Resistance of human cells to tumorigenesis induced by cloned transforming genes.
The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.
Animals↗
Genetic analysis of tumorigenesis: X. Chromosome studies of transformed mutants and tumor-derived CHEF/18 cells.
Chinese hamster embryo fibroblast cell line CHEF/18 is stably diploid, anchorage-dependent, has a high serum requirement, and a does not form tumors in nude mice. The chromosome constitutions of spontaneous and chemically induced anchorage-independent and/or low-serum CHEF/18 mutants and tumors produced in nude mice by some of these mutants are compared. We find a correlation between diploidy and nontumorigenicity among the anchorage-independent mutants but not in the low-serum mutants. One of the four spontaneous and six of the 15 chemically induced anchorage mutants have remained diploid. The remaining 12 mutants are pseudodiploid or aneuploid, and seven of them contain changes in chromosome 1, either a translocation or a deletion involving breakage at the same position (1q11-12). Each of the tumors induced by six mutants has a unique pattern of rearrangements; however five of the six have changes involving chromosome 3. This chromosome was also frequency rearranged in tumor-derived cells previously investigated.
Aneuploidy↗
Genetic analysis of tumorigenesis: XII. Genetic control of the anchorage requirement in CHEF cells.
Chinese hamster somatic cell hybrids between diploid anchorage-independent CHEF/204Bu50 cells and diploid anchorage dependent CHEF/205-30 cells are anchorage dependent but can segregate subclones at low frequency which reexpress anchorage independence. Thus, anchorage independence, like other characteristics of the transformed phenotype, is suppressed in these hybrids. Anchorage-independent subclones were recovered from the anchorage-dependent hybrids under conditions which favored the retention of most chromosomes. Karyotype analysis of suppressed hybrids and their anchorage-independent subclones showed that segregation of anchorage dependence was correlated with the loss of one copy of chromosome 1 in CHEF Chinese hamster hybrids. Thus, suppression of anchorage independence has a chromosomal basis. Several genetic models are considered for the origin of anchorage-independent subclones from suppressed Chinese hamster hybrids.
Animals↗
Tissue specificity and clustering of methylated cystosines in bovine satellite I DNA.
The positions of all 5-methylcytosine (mC) residues in bovine satellite I DNA were determined by sequence analysis of native purified satellite I DNAs from three bovine tissues as well as from cloned DNA. The EcoRI cleavage units from thymus and liver were found to contain 1,402 residues; that from brain contained 1,401 residues. Satellite I DNA from thymus contained a total of 5.0% mC, whereas that from liver and brain contained 4.4% and 2.6% mC, respectively. Thus, the extent of methylation of this DNA is tissue-specific. So is the location. In each tissue, the location of mCs is nonrandom, consisting of three clusters of heavily methylated regions, each of about 200 bases. However, the extent of methylation within each cluster is tissue-specific. The mCs are located entirely in C-G doublets and primarily in palindromic sequences, C-C-G-G sequences (10 methylatable sites) are almost completely methylated in all tissues examined, but T-G-G-A sequences (16 methylated in all tissues examined, but T-G-G-A sequences (16 metylatable sites) are methylated to different extents in each tissue. Neither the tissue specificity of methylation nor the clustering pattern is detectable by examining only G-C-G-G sites, leading us to emphasize the importance of total sequence determination for genomic DNAs in studies of methylation. The clustering pattern, which is preserved despite a 2-fold difference in mC content between brain and thymus, may indicate a role for DNA methylation in chromatin structure.
5-Methylcytosine↗
Pre-adipocyte determination either by insulin or by 5-azacytidine.
CHEF/18 is a diploid Chinese hamster cell line of embryonic origin, which is fibroblastic in structure, but behaves like a mesenchymal stem cell line in its ability to differentiate into adipocytes, myoblasts, and chondrocytes. With these cells, adipocyte formation has been divided experimentally into two stages: (i) determination of pre-adipocytes, which have lost the ability to form other cell types while retaining their fibroblast structure; and (ii) commitment or terminal differentiation, in which lipids accumulate, adipocyte structure develops, and cells lose the ability to divide. This paper reports that the first stage can be induced by exposure to 5-azacytidine or 2'-deoxy-5-azacytidine, drugs that also induce CHEF cells to form other mesenchymal cell types, or by growth with added insulin. Pre-adipocytes are distinguished from CHEF stem cells by (i) their inability to form other mesenchymal cell types; and (ii) their rapid accumulation of lipid in response to added insulin. The possibility is discussed that both insulin and the cytidine analogs promote differentiation by the same mechanism, namely changes in the pattern of DNA methylation.
Adipose Tissue↗