DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture.

T L Kautiainen,P A Jones

doi:10.1016/s0021-9258(17)35981-1

Abstract

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.

Highlights

The levels of DNA methyltransferase in nuclei from 95% (Wigler et al, 1981), and this lack of complete fidelity
Higher concentrations of 5-aza-dCyd were required to be incorporated into theDNA of tumorigenic cells to inhibit DNA methylation by equivalent amounts
These 2 independent methods clearly showed that tumorigenic cells maintained higher methylation capacities than nontumorigenic cells

Summary

MATERIALS AND METHODS

Transformation may involve alterationsin gene expression leading to cancer, it is important to establish whether DNA. The cells present in thesupernatants from the 3 digestions were pelleted by centrifugation and washed in PBS, followed by resuspension in Eagle's minimum essential medium supplemented with 10%heat-inactivated fetal calf serum. These cells were used from passages 2 through 6. Serum Co., Denver, CUI, The C3H/107'1/2 CL8 line (Reznikoff et al, 1973af was used between the 11th and 35th passages and MCA C115 lines (Reznikoff et al, 1973b) were used between the 8th and 24th passages These cells were grown in Eagle's basal medium (Gibcof,supplemented with penicillin and streptomycin (100units/ml) ( G i b ) , and 10% heatinactivated fetalcalf serum. Hepatoma cells (Fao-1;Deschatrette and Weiss, 1974) were grown in equal mixtures of Dulbecco's minimum essential medium and F12 medium (Gibco),supplemented with 10%heat-inactivated fetalcalf serum. DNA content was measured using the fluorometric method of Kissane and Robins (1956)

RESULTS

ACZdAR

DISCUSSION