The pituitary transforming gene, PTTG, is abundantly expressed in endocrine neoplasms. PTTG has recently been recognized as a mammalian securin based on its biochemical homology to Pds1p. PTTG expression and intracellular localization were therefore studied during the cell cycle in human placental JEG-3 cells. PTTG mRNA and protein expressions were low at the G₁/S border, gradually increased during S phase, and peaked at G₂/M, but PTTG levels were attenuated as cells entered G₁. In interphase cells, wild-type PTTG, an epitope-tagged PTTG, and a PTTG-EGFP conjugate all localized to both the nucleus and cytoplasm, but in mitotic cells, PTTG was not observed in the chromosome region. PTTG-EGFP colocalized with mitotic spindles in early mitosis and was degraded in anaphase. Intracellular fates of PTTG-EGFP and a conjugate of EGFP and a mutant inactivated PTTG devoid of an SH3-binding domain were observed by real-time visualization of the EGFP conjugates in live cells. The same cells were continuously observed as they progressed from G₁/S border to S, G₂/M, and G₁. Most cells (67%) expressing PTTG-EGFP died by apoptosis, and few cells (4%) expressing PTTG-EGFP divided, whereas those expressing mutant PTTG-EGFP divided. PTTG-EGFP, as well as the mutant PTTG-EGFP, disappeared after cells divided. The results show that PTTG expression and localization are cell cycle-dependent and demonstrate that PTTG regulates endocrine tumor cell division and survival.

Pituitary tumor transforming gene (PTTG) was identified in rat pituitary tumor cells by differential mRNA display (1). Human PTTG (2, 3) is highly expressed in pituitary tumors (4, 5) and other neoplasms (3, 6, 7). Levels of PTTG expression positively correlate with pituitary tumor invasiveness (4) and are induced by estrogen (5). The mechanism of PTTG action is not clear. PTTG up-regulates basic fibroblast growth factor secretion (2) and transactivates DNA transcription (Refs. 3, 8 and Horwitz, G. A., Z. Wong, X. Zhang, and S. Melmed, unpublished). Recently, PTTG protein has been recognized as a mammalian securin protein that maintains binding of sister chromatids during mitosis (10). At the end of metaphase, securin is degraded by an anaphasepromoting complex, releasing tonic inhibition of separin, which in turn mediates degradation of cohesins, the proteins that hold sister chromatids together. Overexpression of a nondegradable PTTG disrupts sister chromatid separation (10).

Since PTTG was identified, its cellular characteristics have not been studied. To address the mechanism of PTTG action, we studied PTTG expression and intracellular localization during the cell cycle in human placental JEG-3 cells, which are among the few available human endocrine cells in culture. The effects of PTTG expression on the cell cycle were also addressed. JEG-3 cells secrete hCG both basally and in response to a variety of stimulants. Our results show that PTTG expression and localization are cell cycle dependent and demonstrate that PTTG regulates placental tumor cell division and survival.

Cell Cycle-Dependent PTTG Expression

Human placental JEG-3 cells express PTTG (Fig. 1). After JEG-3 cells were synchronized at the G₁/S border with double thymidine block and then released, they gradually entered S, G₂/M, and G₁ over 15 h (Fig. 1A). JEG-3 cells were also synchronized at the G₁/S border and at G₂/M by treating with aphidicolin and nocodazole, respectively (Fig. 1A). PTTG mRNA expression was low at the G₁/S border achieved by double thymidine block or by incubation with aphidicolin (Fig. 1B). When cells were released, PTTG mRNA increased through the S phase and plateaued at G₂/M (2.0 ± 0.3-fold over G₁/S). Cells synchronized by nocodazole also expressed higher mRNA levels than did cycling cells. PTTG protein expression followed a similar cycle-dependent course (Fig. 1C). PTTG protein levels assessed by Western blotting were very low in cells at the G₁/S border, increased through S phase, and peaked at G₂/M (2.4 ± 0.6-fold over G₁/S). When cells divided, PTTG protein levels again decreased.

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