Expression of the hypoxanthine phosphoribosyl transferase gene in resting and growth-stimulated human lymphocytes.
Expression of the hypoxanthine phosphoribosyl transferase (hprt) gene was studied in resting and growth stimulated human lymphocytes by determinations of hprt-RNA and enzyme activity levels in cell extracts. Hprt-RNA was determined by quantitative solution hybridization and enzyme activity by measuring the rate of conversion of [14C]hypoxanthine to inosine 5'-monophosphate. In resting Go-lymphocytes, the hprt activity (5 nmol/h per 10(6) cells) was twice as high as in human fibroblasts, whereas the hprt-RNA level was very low (0.3 pg per 10(6) cells, or approx. one mRNA molecule per cell). Both RNA and enzyme activity remained at these levels in cells that were incubated in the presence of cycloheximide (CHX, 20 micrograms/ml) for up to 48 h, which suggests that hprt gene expression in resting lymphocytes depends mainly on a stable protein with a half-life of more than 48 h. In phytohemagglutinin (PHA) stimulated lymphocytes, the hprt-RNA levels increased 10-20-fold, while the enzyme activity increased 5-fold. The addition of hydroxyurea (0.01 M) to the culture medium did not prevent the increase of hrpt-RNA, whereas the increase of both RNA and enzyme activity was abrogated in the presence of CHX. Thus, the induction of hprt expression in growth stimulated lymphocytes requires new protein synthesis, and is not coupled to DNA replication. In long-term lymphocyte cultures, both hprt-RNA and enzyme activities showed high steady-state levels. After removal of PHA and growth factors from the culture medium the hprt-RNA levels decreased by over 80% within 24 h, while the enzyme activity was unaffected. Inhibition of transcription by actinomycin D (5 micrograms/ml) caused a rapid decay of the hprt-RNA, with an estimated half-time of 5.1 h. When CHX was added to actinomycin D inhibited cells, the rate of hprt-RNA breakdown was reduced. The hprt enzyme activity declined by approx. 50% during 24 in the presence of CHX. Thus, the enhanced expression of hprt-RNA in proliferating lymphocytes depends on continuous growth stimulation and seems to be associated with a high transcriptional activity and turnover of the RNA. In contrast, the enzyme activity is relatively stable, and less sensitive to alterations of growth conditions.
Department of Clinical Genetics, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
A M
AM
Steen
S
S
Sahlén
B
B
Lambert
D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology*; D003609:Dactinomycin / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009693:Nucleic Acid Hybridization; D010835:Phytohemagglutinins / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000032:analysis; D012323:RNA Processing, Post-Transcriptional
D010835:Phytohemagglutinins; D003609:Dactinomycin; D012313:RNA; D003513:Cycloheximide; D007041:Hypoxanthine Phosphoribosyltransferase