Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes.
ABSTRACT IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF α and exposure to TGF β inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.
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ABSTRACT: A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.Proceedings of the National Academy of Sciences 04/1989; 86(5):1578-82. · 9.74 Impact Factor
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ABSTRACT: A new technique for assaying infectivity of adenovirus 5 DNA has been developed. Viral DNA was diluted in isotonic saline containing phosphate at a low concentration, and calcium chloride was added, resulting in the formation of a calcium phosphate precipitate. DNA coprecipitated with the calcium phosphate and, when the resulting suspension was added to human KB cell monolayers, became adsorbed to the cells. Following adsorption, uptake of DNA into the cells occurred during an incubation in liquid medium at 37 ° in the continued presence of extra calcium chloride.For adenovirus 5 DNA the assay resulted in up to 100-fold more plaques than could be obtained using DEAE-dextran. Furthermore a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated. The assay was most efficient at high DNA concentrations (10–30 μg/ml); below this range the addition of carrier DNA was necessary for optimum results.In addition to adenovirus 5 DNA, the technique has been used successfully to assay infectivity of DNA from adenovirus 1 and simian virus 40.Virology 05/1973; 52(2):456-67. · 3.37 Impact Factor
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ABSTRACT: Two epithelial cell lines have been established from the duodenum (IEC-17) and the ileum (IEC-18) of outbred germfree Crl:CD(SD)GN rats. They have a very similar morphology and ultrastructure, a normal rat diploid karyotype, comparable growth rates, and a similar set of surface antigens as detected with monoclonal antibodies specific for intestinal epithelial cell surface proteins in vivo. Both cell lines do not grow in soft agar and when injected into syngeneic animals do not form tumors. The toxic effects of benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) were studied in these two cell lines. The IEC-18 cells were found to be much more sensitive to both chemicals than the IEC-17 cells. In particular, at concentrations greater than 1 microgram/ml, DMBA completely and irreversibly inhibited cell proliferation in the IEC-18 cells, whereas it produced a less marked and reversible inhibition in the IEC-17 cells. The metabolism of BP and DMBA to water-soluble products was studied; BP was apparently metabolized at similar rates in the two cell lines, whereas a striking difference was observed with DMBA, which was metabolized at a rate 10-15 times greater in the IEC-18 cells than that in the IEC-17 cells. These results suggest that cultured cell lines derived from different portions of the intestinal tract have similar properties in vitro, but they may have very different responses to chemical carcinogens.JNCI Journal of the National Cancer Institute 01/1982; 67(6):1353-62. · 14.34 Impact Factor