PTTG has a dual role in tumorigenesis. Firstly, it regulates expression of basic fibroblast growth factor (FGF-2), and secondly, as a human securin homologue, it inhibits sister chromatid separation in mitosis. Securins must be degraded at Destruction Box (DB) motifs by an anaphase promoting complex (APC), before cell division can proceed. We have used a variety of approaches to examine the precise roles of the PTTG Destruction Box and FGF-2 stimulation in cell transformation. PTTG was cloned into a mammalian expression vector and the DB consensus disrupted by amino acid substitutions R60K, L63V and G64A. NIH-3T3 cells were stably transfected with control vector, wild type PTTG and DB mutants. 10,000 cells were seeded per dish in soft agar and transforming ability assessed after two weeks. Overexpression of control cell lines failed to elicit transformation [27 plus/minus 7 colonies/well], whereas wild type PTTG transfectants yielded large colonies [183 plus/minus 17 colonies/well]. DB mutants, however, were incapable of producing colonies in 3T3 cells [21 plus/minus 4 colonies/well]. Since PTTG regulates FGF-2, we assessed mRNA expression of this gene in stable transfectants. Northern analysis demonstrated identical FGF-2 expression. Further, in transiently transfected MCF-7 cells, wild type and DB mutants exhibited indistinguishable stimulation of FGF-2 (2.0-fold and 1.9-fold respectively, compared with control), indicating that cell transformation is not dependent upon PTTG-mediated FGF-2 upregulation. Sequencing of the DB in 74 sporadic pituitary tumours revealed no changes from wild type. An EGFP-tagged DB construct transfected into MCF-7 cells demonstrated raised intensity of nuclear staining compared with wild type EGFP-PTTG, suggesting increased protein stability within the nucleus. Conclusions: Destruction Box mutations, which may increase PTTG stability, are not a common cause of pituitary tumours. Integrity of the DB is required for PTTG-mediated cell transformation, but not FGF-2 stimulation. In vivo, FGF-2 upregulation may therefore promote mitogenesis and angiogenesis in already-transformed cells.
08 - 11 Apr 2002
British Endocrine Societies