Cell cycle- and age-dependent transcriptional regulation of human thymidine kinase gene: the role of NF-Y in the CBP/tk binding complex.
Expression of thymidine kinase gene in normal human diploid cells is both cell cycle- and age-dependent and appears to be transcriptionally regulated. Strong DNA protein binding with a 28-bp fragment (-91/-64) that contains the distal inverted CCAAT box is observed in serum-stimulated young (low population doubling level) IMR-90 cells but not in senescent cells. This cell cycle- and age-dependent binding factor was termed CBP/ tk, indicating CCAAT binding protein for the thymidine kinase gene. Based on immunoshift assay and purification, it has been suggested that CBP/tk is equivalent to NF-Y, previously identified as the binding protein for the Y box within E alpha gene promoter. In this study, we examined the mRNA level and protein amount of NF-Y, proteins in young and old IMR-90 cells during serum stimulation by Northern and Western blot blot analyses. In addition, we compared (1) the turnover rate of NF-Y in IMR-90 cells with that of CBP/tk binding activity and (2) the levels of NF-Y and CBP/tk in normal and cancer cells. Both NF-YA and NF-YB were constitutively expressed at mRNA level in IMR-90 cells. However, expression of NF-YA, and to a lesser degree, NF-YB, at the protein level were clearly age-dependent. The half-life of NF-YA and NF-YB were, respectively, 4- and > 10-fold longer than that of CBP/tk binding activity in IMR-90 cells. In addition, we found that the amount of NF-Y did not correlate with the overexpression of CBP/tk binding activity in HeLa cells. Taken together, our results suggested that although CBP/tk may contain NF-YA or related proteins, NF-A and NF-YB alone may not account for all the characteristics of CBP/tk observed in normal and transformed human cells.
Good
L F
LF
Department of Chemistry, Rutgers-The State University of New Jersey, Piscataway 08855-0939, USA.
Chen
K Y
KY
D000256:Adenoviridae / Q000235:genetics; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D002453:Cell Cycle; D002460:Cell Line; D016922:Cellular Senescence; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II; D006207:Half-Life; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D013937:Thymidine Kinase