Recent advances in the study of rTS proteins. rTS expression during growth and in response to thymidylate synthase inhibitors in human tumor cells

The rTS proteins have now been shown to be expressed in a variety of cell lines, with expression of rTSβ being found elevated in three cell lines which are resistant to TS inhibitors (3,4) (Figure 1). In one of these cell lines (K562 B1A), the cells were selected for resistance to MTX, which has a primary site of action on DHFR, but was found to be cross-resistant to FUdR (4). The other two cell lines were selected for resistance to either 5-fluorouracil (H630-1) or a combination of ZD1694 and FU. In each case, elevation of rTSβ appears to be a selected response to thymidylate stress. In HCT-8 and HCT-8/DF2 cells, treatment of cells for a short period of time (2 hr) resulted in the elevation of rTSβ levels, again suggestive that expression of rTSβ is a response to thymidylate stress. rTSβ appears to be regulated with cell growth, its levels increasing at mid-log and at late-log/saturation phase in H630 and H630-1 cells (Fig. 2), and increasing with late-log in several other cell lines as well (Fig. 3). The increase in rTSβ is suggestive of a cellular function associated with a state where growth is no longer desirable, reminiscent of the starvation-sensing protein homolog RSPA in E. coli (22). While this relationship would not explain the spike in rTSβ levels in mid-log H630 and H630-1 cells, it does make sense if the rTS proteins (particularly rTSβ) are involved in down-regulating thymidylate biosynthesis. The potential mechanism of this down-regulation may be speculated to be the catabolism of some precursor for thymidylate biosynthesis or some direct effect upon TS through modulation by some other ligand, either a metabolite or another protein. Studies on the expression of rTS proteins in clinical specimens indicate that rTSβ is expressed at high levels in kidney and kidney tumor (Dolnick, unpublished results). Given the physiologic role of the kidney, high level expression of rTS in this organ is consistent with a role in a catabolic pathway. Since down-regulation of TS activity is expected to increase sensitivity to TS inhibitors, a role for rTSβ in directly down-regulating TS activity in the biochemical sense would seem unlikely. However, the manner of biochemical TS down-regulation may make a difference. In the TS− C1/C1 cell line, there are two mutations in TS which likely reduce affinity for N-5,10-methylene tetrahydrofolates (23). This cell line is highly resistant to MTX, yet is still tumorigenic in vivo (24), and supplying the cells with high levels of exogenous folate can restore TS function (23). Thus in TS− C1/C1 cells, the TS phenotype is conditionally dependent upon the presence of high levels of exogenous folate. This suggests that a role of rTS proteins as conditional down-regulators of TS, perhaps through modulating folate binding, may be possible. Two cell lines (K562 B1A and H630-1) that overproduce rTSβ have altered sensitivity to TS inhibitors that differ depending upon the nature of the inhibitor. The K562 B1A cell line was found to be approximately 2000-fold resistant to ZD1694 and BW1843U89 (120 hr exposures∗), but only threefold resistant to AG331. The H630-1 cell line is approximately 30-fold resistant to BW 1843U89 (120 hr exposure) and 40-fold resistant to ZD1694 (120 hr exposure), but only eight-fold resistant to AG331†. Since K562 B1A cells overproduce rTSβ (2), but have no significant alterations in FPGS activity, the possibility that rTS may affect folate binding remains a hypothesis worth examining. The recent discovery that TS is a phosphoprotein and that it is nuclear as well as cytoplasmic (21) raises the possibility that the phosphorylation state of TS may regulate one of its cellular functions, and that the subcellular localization of this enzyme is regulated as well. Since rTS proteins have HSP with proteins that participate in kinase/phosphatase reactions, this also seems to be an avenue worthy of future investigation. In summary, while the expression of rTS proteins continues to suggest an effect on TS function, the precise function of rTS has yet to be established but the observations discussed here suggest several areas for future study.