Determination of queuosine modification system deficiencies in cultured human cells

Queuosine-deficient tRNAs are often observed in neoplastic cells. In order to determine possible sites for malfunction of the multistep queuosine modi fication system, comprehensive studies were performed on two human neoplast ic cell lines, the HxGC(3), colon adenocarcinoma and the MCF-7 breast adeno carcinoma, which are 100 and 50-60% queuosine deficient, respectively. Thes e results were compared with data obtained hom normal human fibroblast (HFF ) cultures which maintain 100% queuosine-modified tRNA populations. Queuine uptake in all three cell types was similar and each demonstrated activatio n by protein kinase C (PKC). However, incorporation of queuine into tRNA by tRNA:guanine ribosyltransferase (TGRase; E.C. 2.4.2.24) and PKC catalyzed activation of this enzyme occurred only in HFF and MCF-7 cells. The HxGC(3) , cell line exhibited no TG;Rase activity as was expected. Treatment with 5 -azacytidine (5-azaC) induced TGRase activity to a level 20% of that in HFF and MCF-7 cells; however, this 5-azaC-induced TGRase activity was not regu lated by PKC. Salvage of the queuine base from tRNA degradation products ha s been shown in mammalian cells and was measured in the HFF cells. However, salvage activity in the MCF-7 cell line was deficient. Therefore, it was s hown by direct measurements that the HxGC(3) cell line is completely lackin g in queuosine-modified tRNA due 60 loss of functional TGRase, while the MC F-7 cell line has an inefficient queuine salvage mechanism resulting in a s ignificant deficiency of queuosine-modified tRNA. These techniques can be a pplied to any cultured cell types to determine specific lesions of the queu osine modification system, which have been suggested to be associated with neoplastic-progression. (C) 1999 Academic Press.