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Determination of queuosine modification system deficiencies in cultured human cells
R C Morris1, M C Galicia, K L Clase
1Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529-0126, USA.
Abstract:
Queuosine-deficient tRNAs are often observed in neoplastic cells. In order to determine possible sites for malfunction of the multistep queuosine modification system, comprehensive studies were performed on two human neoplastic cell lines, the HxGC(3) colon adenocarcinoma and the MCF-7 breast adenocarcinoma, which are 100 and 50-60% queuosine deficient, respectively. These results were compared with data obtained from normal human fibroblast (HFF) cultures which maintain 100% queuosine-modified tRNA populations. Queuine uptake in all three cell types was similar and each demonstrated activation 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 TGRase 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 regulated by PKC. Salvage of the queuine base from tRNA degradation products has 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 shown by direct measurements that the HxGC(3) cell line is completely lacking in queuosine-modified tRNA due to loss of functional TGRase, while the MCF-7 cell line has an inefficient queuine salvage mechanism resulting in a significant deficiency of queuosine-modified tRNA. These techniques can be applied to any cultured cell types to determine specific lesions of the queuosine modification system, which have been suggested to be associated with neoplastic progression.
Insights
Cancer cells often lack queuosine-modified tRNA due to defects in the queuosine modification system. This study identifies a complete loss of tRNA guanine ribosyltransferase (TGRase) in colon cancer cells and deficient salvage mechanisms in breast cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Queuosine-modified tRNAs are crucial for cellular function and are often deficient in neoplastic cells.
- Understanding defects in the queuosine modification pathway is vital for cancer research.
Purpose of the Study:
- To investigate the molecular basis of queuosine deficiency in human colon (HxGC(3)) and breast (MCF-7) adenocarcinoma cell lines.
- To compare the queuosine modification system in cancer cells with normal human fibroblasts (HFF).
Main Methods:
- Assessed queuine uptake and activation by protein kinase C (PKC) in HxGC(3), MCF-7, and HFF cells.
- Measured tRNA:guanine ribosyltransferase (TGRase) activity and its regulation by PKC.
- Evaluated the salvage of queuine base from tRNA degradation products.
Main Results:
- HxGC(3) cells completely lack functional TGRase activity.
- MCF-7 cells exhibit deficient queuine salvage mechanisms, leading to significant queuosine deficiency.
- Both cell lines showed similar queuine uptake and PKC activation, but TGRase incorporation differed.
Conclusions:
- The HxGC(3) colon cancer cell line's queuosine deficiency stems from a complete loss of TGRase.
- The MCF-7 breast cancer cell line's deficiency is due to an inefficient queuine salvage pathway.
- These findings highlight specific defects in the queuosine modification system associated with cancer progression.