Nuclear Expression of Thymidylate Synthase in Colorectal Cancer Cell Lines and Clinical Samples

Seema Bissoon-Haqqani ¹, Terence Moyana ¹, Derek Jonker ¹, Jean A. Maroun ¹ and H. Chaim Birnboim ^1*

¹ Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada (SB-H,HCB); Department of Laboratory Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada (TM,HCB); and Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada (DJ,JAM,HCB)

^* To whom correspondence should be addressed. E-mail: birnboim{at}uottawa.ca.

Submitted on February 3, 2005
Accepted on 11 May 2005

	Abstract

Thymidylate synthase (TS) (TYMS; OMIM reference number (188,350)) is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS was also seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-FUdR were fractionated and examined by Western blotting. Interestingly, both free TS and the ‘ternary complex’ of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS and HT-29, RKO and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence (NES) was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.

Key Words: thymidylate synthase, colorectal cancer, nuclear, immunohistochemistry

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