Genetic analysis of tumorigenesis: V. Chromosomal analysis of tumorigenic and nontumorigenic diploid chinese hamster cell lines

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-Haras 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-rasl 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 EJcontaining 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.

Section: Discussionmentioning

confidence: 87%

Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.

Lau¹,

Gadi²,

Kalvonjian³

et al. 1985

“…The latter cell line contains a homogeneous staining region on chromosome 3 (23). It was of interest to see whether we could detect a difference in the m5Cyt distribution in DNAs from these two cell lines.…”

Section: Methodsmentioning

confidence: 99%

Identification of 5-methylcytosine in DNA fragments immobilized on nitrocellulose paper.

Sano¹,

Royer²,

Sager³

1980

A method to identify in DNA immobilized on nitrocellulose paper by using antibody against m5Cyt raised in rabbits is described. Immobilized restriction fragments of DNA are incubated first with purified antibody against m5Cyt and then with goat anti-rabbit IgG labeled with 125I. Restriction The only known post-replicative modification of eukaryotic DNA is methylation of cytosine residues at the 5-position (1). A function for 5-methylcytosine (m5Cyt) in eukaryotic gene regulation, first proposed by Scarano (2), has been suggested in several recent studies (3, 4).*In Chlamydomonas, we have demonstrated that the maternal inheritance of chloroplast DNA is regulated by a restriction modification system analogous to that in bacteria (5).Methods have been developed to identify m5Cyt, including paper and liquid chromatography (6-8), two-dimensional electrophoresis (9), and mass spectrometery (10); they identify and quantify m5Cyt but provide no information on its location. Precise sequence localization can be established by the method of Maxam and Gilbert (11) but only with uncloned DNA, because sequences cloned in bacteria are not methylated at the uncloned methylation sites. By using pairs of isoschizomeric endonucleases such as Msp I which cleaves C-mCG-G and Hpa II which cleaves only the unmethylated C-C-G-G, methylated C-C-G-G sequences can be localized to particular restriction sites (12). However, there are additional methylated sites for which pairs of isoschizomeric nucleases are not available-for instance, as revealed by direct sequence determination of 5S rDNA (13). An immunological method used to identify m5Cyt in chromosome preparations showed localized regions of heavy DNA methylation at the centromeres (14) but was not applied to the molecular level.This paper describes a method to identify m5Cyt in DNA immobilized on nitrocellulose paper by using antibody raised against m5'Cyt. With this method, potentially every methylated cytosine in DNA can be detected. MATERIALS AND METHODSAntibody Preparation. A 100-mg sample of 5-methylcytidine was conjugated to 280 mg of bovine serum albumin (15) and 1 mg with complete Freund's adjuvant was injected into each rabbit at 2-week intervals (16). When the precipitin test became positive, 50-ml blood samples were taken, and antibody against m5Cyt was prepared from serum as described (16) using m5Cyt-bovine serum albumin-conjugated Sepharose 4B. Antiserum to rabbit IgG produced in goats were purified by using rabbit immunoglobulin-conjugated Sepharose 4B. Antibody preparations were iodinated as described (17). The iodinated protein was fractionated by Sephadex G-50 column chromatography and purified further by three cycles of precipitation with 40% ammonium sulfate. The final pellet was dissolved in buffer I (10 mM K phosphate, pH 7.1/0.15 M NaCl) and stored at -20°C. The specific activity was 106 cpm/jg when prepared. Filter Binding Assay. Sonicated and denatured DNAs from calf thymus and Escherichia coli were labeled at the 5' end with [,y-2P]ATP by polynu...

“…At confluence, the medium was removed and replaced with low serum medium (i.e., 2.0% FCS-DME medium); cells were fed every 3rd day thereafter. After 14-21 days, foci corresponding to transformed phenotypes were scored under a dissecting microscope as described (16,17). Transformed colonies were selected for (i) ability to grow in medium containing low serum, (ii) loss of contact inhibition (i.e., cells piled on one another in foci), and (iii) transformed morphology corresponding to previously described tumor-forming phenotypes (16,17).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of radiation-induced neoplastic transformation by beta-lapachone.

Boothman

Pardee

1989