Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite,are effective chemopreventive agents for various chemically induced tumorsin animal models at both the initiation and postinitiation stages. Themechanisms involved at the postinitiation stage are not clear. Becauseseveral lines of evidence indicate that inhibition of excess DNA(cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for thesuppression or reversion of carcinogenesis, we examined the effects ofsodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analogof BSC, on Mtase activity in nuclear extracts of human colon carcinomas,and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells inculture. For this purpose, we developed an improved Mtase assay, in whichthe incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), isspecifically determined by HPLC with radioflow detection after enzymatichydrolysis, enhancing specificity and reliability. In a variation, usingSssI methyltransferase and labeled S- adenosylmethionine, the overallmethylation status of DNA in various tissues can also be compared.Selenite, BSC and p-XSC inhibited Mtase extracted from a human coloncarcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had noeffect. p-XSC also inhibited the Mtase activity and growth of human coloncarcinoma HCT116 cells, with an IC50 of approximately 20 microM. Theimproved Mtase assay should prove to be a reliable method for screeningpotential Mtase inhibitors, especially using cells in culture. We suggestthat inhibition of Mtase may be a major mechanism of chemoprevention byselenium compounds at the postinitiation stage of carcinogenesis.