An investigation of thymidine kinase 1 from normal and transformed mammary cell lines. FIONA BRITTON , BIRGITTE MUNCH-PETERSEN2,. REINHOLD ...
Biochemical Society Transactions (1998) 26 S63 72
An investigation of thymidine kinase 1 from normal and transformed mammary cell lines.
FIONA BRITTON , BIRGITTE MUNCH-PETERSEN2, REINHOLD HOFBAUER3 and YVONNE A. BARNETTI’. ‘Cancer and Ageing Research Group, University of Ulster, Coleraine, BT52 ISA, Northern Ireland. 2Institute of Life Sciences and Chemistry, Roskilde University, DK-4000, Denmark Institute of Molecular Biology, Vienna Biocenter, University of Vienna, A-1030, Austria. Thymidine kinase 1 (TKl; EC 2.7.1.21) catalyses the phosphorylation of thymidine to form thymidine monophosphate (dTMP). Although work on established mammalian cell lines suggests that TKI may be important to ensure the accuracy of DNA repair processes [e.g., I], the precise biological function of TKI in vivo remains to be elucidated. TKI activity is cell cycle regulated; transcriptional, post-transcriptional and translational activation of TKl expression lead to elevation of activity at the GI/S phase [2] and degradation in M phase [3]. An additional regulatory mechanism for TKl has been found, which operates at the enzymatic level and controls an ATP-dependentreversible shift between a low and high affinity form of TKI. The form of TKl catalysing the phosphorylation of thymidine depends on the concentration of enzyme and pre-exposure to ATP. The preexposure enables the transition from the dimer to the tetramer [4]. In this investigation we examined; TKl protein levels and activity, and the effect of ATP on TKI activity extracted from exponentially growing normal (HBL- 100) [ 5 ] and transformed mammary cell lines (MCF-7 [6], ZR75-1 [7] and T47-D [8]).All cell lines had similar generation times (22-25 hours). Cell lysates were processed to obtain a soluble source of TK1 for the activity assays. 1x106 cells were lysed in buffer (20mM Tris pH 7.8, 200mM KCI, 5mM MgC12. 5mM B-mercaptoethanol, 1% NP-40). To assess the effect of ATP on TKl activity 2mM ATP was added to this buffer. Cells were disrupted by freeze-thawing (x3) and cell debris was removed by centrifugation (2oooOg, 30 min at 4oC). The supernatant was used as a source of TKl. Protein concentrations were measured using the Biorad Protein Assay. TKI activity in the +/- ATP extracts was determined using the assay described by McKenna et al. [9]. The assay buffer consisted of 20mM Tris pH 7.8, 200mM KCI, lOOmM NH4CI, 2mM MgC12, 0.5 mg/ml BSA, 5mM B-mercaptoethanol, 2mM ATP (only for those cell extracts prepared in the absence of ATP) and 2mM [3H]-methyl thymidine. Equal volumes of cell lysate and assay buffer were mixed and incubated at 37.C. After 30 min, 2 5 ~ aliquots 1 were spotted onto DEAE-81 discs and then washed in 0.1M ammonium formate ( ~ 3 )dHIO . (XI) and ethanol (xl). The radioactivity ([3H] dTMP) on the discs was determined by scintillometry. Western blot analysis of mammary cell extracts was performed to assess TKl protein levels. Cell lysates containing 100pg protein were loaded onto 14% SDS-PAGE gels and protein separation achieved by electrophoresis at 40 mA, 50V, overnight at 4.C. Following electroblotting onto nitocellulose membranes probing with rabbit anti-human TK1-specific polyclonal antibody (2 hours at 4 C ) was performed. After washing, sheep anti-rabbit IgG antibody coupled to peroxidase was incubated with the membrane (2 hours at 4oC). Visualisation of the bands was achieved using ECLTM chemiluminescent solution (Amersham) and autoradiography. Following Western blot analysis scanning densitometry of the 24 kDA (TKI) band, which appeared in extracts from all cell lines, revealed much more TKI protein in extracts from MCF-7 (-6x) and ZR75-1 cells (-2Ox) than was present in HBL-100 and T47-D cells. In the absence of ATP, the amount of active TKI present was higher in MCF-7 and ZR75-1 cells, than in HBL-100 and T47-D cells (Figure 1). *Correspondingauthor
HBL-I00
MCF-7
ZR75-1
2175-30
T47-D
Mammary Cell Lines
Figure 1: TK1 activity and the effect of ATP on TK1 activity in the mammary cell lines. The results shown are the mean SEM of three independent experiments.
*
The presence of ATP in the extraction buffer increased TKI activity from all cell lines. However, the extent of the increase was less in extracts from MCF-7 ( 1 . 8 ~ )and ZR75-1 ( 1 . 4 1 ~ )than HBL-100 ( 2 . 9 ~or ) T47-D (3.05~)cells (Figure 1). These results suggest that TKI protein production and regulation of activity by ATP is altered in the transformed mammary cell lines, MCF-7 and ZR75-1, when compared to the normal mammary cell line (HBL-100) and the transformed mammary cell line (T-47D), with more active TK1 being produced, perhaps in an attempt to compensate for the alterations to the control of TKI activity exerted by ATP. The biological significance of the altered TKI and the molecular process(es) leading to the alterations in MCF-7 and ZR75-1 cells are currently under investigation.
1. McKenna, P.G., Wilkinson, Y.A. and McKelvey, V.J. (1991) Anticarcinogen. Radiat. Protec. 2.73-77 2. Sherley,-J.L. and Kelly, T.J. (1988) J. Biol. Chem. 263, 8350-8358 3. Kauffman, M.G. and Kelly , T.J. (1991) Mol. Cell. Biol. 11, 2358-2546 4. Munch-Petersen, B., Tyrsted, G. and Cloos, L. (1993) J. Biol. Chem. 268, 15621-15625 5 . Gaffney, E.V. (1982) Cell Tissue Res. 227, 563-568 6. Soule, H.D., Vanquez, J., Long, A., Albert, S., Brennan, M. (1973) J. Natl. Cancer Inst. 51, 1409-1416 7. Engel, L.W., Young, N.A., Tralka, T.S., Lippman, M.E., O’Brien, S.J. and Joyce, M.J. (1978) Cancer Res. 38, 33523364 8. Lippman, M.E., Dickson, R.B. and Lupu, R. (1991) in Origins of human cancer: A comprehensive review (Brugge, J., Curran, T., Harlow, E. and McCormick, F., eds.), pp. 645-653, Cold Spring Harbor Laboratory Press, New York 9. McKenna, P.G., O’Neill, K.L., Abram, W.P. and Hannigan, B. M. (1988) Br. J. Cancer 57, 619-622
