Oncogenic function of PTTG1 was established by its overexpression in mouse fibroblast cell line (NIH 3T3) followed by assessment of its ability to induce cellular transformation and tumor formation in nude mice [2, 4]. However, the differences in biology between the rodent cells and human cells have brought the validity of this model into question. There are a number of instances in which an oncogene has been shown to induce transformation in rodent cells but has failed to induce transformation of same types of cells obtained from humans. To test the ability of PTTG1 to induce transformation in human cells, we selected the human embryonic kidney-293 (HEK293) cell line as our model. Our data clearly demonstrate that over expression of PTTG1 in HEK293 results in an increase in cell proliferation induces cellular transformation in-vitro (increase in anchorage-independent growth) and promotes tumor formation in nude mice. Cells transfected with pcDNA3.1 vector did not form colonies in soft agar or develop into tumors on implantation in nude mice, confirming that HEK293 cells do not possess a tumorigenic phenotype. Furthermore our data suggest that overexpression of PTTG1 in these cells results in a greater propensity for tumor development, a shortened latency period and an enhanced growth rate compared with pcDNA3.1-transfected control HEK293 cells.
A second issue that we were able to address using the HEK293 cell model was the question of the ability of PTTG1 to induce transformation of normal human cells, i.e., whether it acts alone or in cooperation with another oncogene to achieve its tumorigenic function. It has been reported that a single oncogene may not be sufficient for induction of transformation but requires co-expression, or oncogenic cooperation, of another oncogene(s) to induce tumorigenesis in normal primary human cells [20, 21, 24, 25, 42, 43]. Our results clearly show that PTTG possess the ability to induce cellular transformation in vitro and promotes tumor formation in nude mice. Since, HEK293 cells are transformed with adenovirus type 5 thus making them different from the normal primary cells, therefore it remains unclear if PTTG is sufficient by itself to initiate the tumorigenesis of primary human cells. However, the data clearly demonstrate that PTTG1 overexpression in these cells accelerates their tumorigenic capacity in comparison to that of unmodified cells.
PTTG1 contains several-proline rich motifs (PXXP); two of these that are located in the C-terminal domain have been reported to be potential binding sites for SH3-domians . In our study we confirm that mutation of these C-terminal proline-rich motifs abrogates the tumorigenicity of PTTG1 in human cells. Such loss of tumorigenicity on mutation of PTTG1 could be due to a loss of expression. Our western blot analysis of the stable cell lines (HEKmPTTG1-2 and HEKmPTTG1-4) that constitutively express mutated PTTG1 protein showed high levels of expression of mPTTG1 protein (Fig. 1), suggesting that the loss of tumorigenic function of mPTTG1 protein is not due to loss of expression but due to the loss of its ability to induce cellular transformation. These results are consistent with other investigators for rodent cells  and confirm the importance of C-terminal proline-rich motifs to mediate the oncogenic function of PTTG1.
The molecular mechanisms by which PTTG1 achieves its tumorigenic function remain unclear. PTTG1 has been reported to induce expression of the c-myc oncogene , bFGF  and VEGF . bFGF is a broad spectrum and pleiotropic mitogen for growth and differentiation affecting various mammalian cells and organ systems and a large number of cells lines [45, 46]; besides stimulating wound healing, tissue repair and hematopoiesis , bFGF induces cell migration and proliferation  and acts as an agiogenic factor that induces migration, proliferation and differentiation of endothelial cells . In addition, it has been reported to modulate the invasion of tumor cells through surrounding tissue to form new capillary cord structures by regulating the activities of extracellular molecules including collagenase, proteinases and integrins . Regulation of secretion and expression of bFGF by PTTG1 in NIH 3T3 cells has been shown . Consistent with these reports, our results demonstrate a significant increase in secretion and expression of bFGF in HEK293 cells on transient transfection with PTTG1 cDNA (Fig. 6) as well as in tumors developed by injection of stable cell lines that constitutively express PTTG1 both at protein and mRNAs levels (Figs. 6, 7, 8).
VEGF is a potent stimulant of the vascularization of tumors and is one of the most specific markers of tumor vasculature observed to date [50, 51]. VEGF is a multifunctional cytokine acting as a potent permeability agent, an endothelial cell chemotactic agent, an endothelial cell survival factor and an endothelial cell proliferation factor . The expression and secretion of VEGF has been shown to be a crucial rate-limiting step during tumor progression . Our results demonstrate a significant increase in secretion and expression of VEGF in HEK293 cells on transfection with PTTG1 and also from tumors excised from animals injected with HEK293 cells that stably express PTTG1 (Figs. 6, 7, 8).
A direct correlation between high IL-8 expression and metastases in melanoma , ovarian cancer , prostrate cancer  and pancreatic cancer  has been reported. To determine if overexpression of PTTG1 induces change in secretion and expression of IL-8, we measured its levels in HEK293 cells on transfection with PTTG1 cDNA and in tumors developed on injection of HEK293 cells transfected with PTTG1. Our results demonstrate for the first time that overexpression of PTTG1 induces IL-8 expression in vitro and also in tumors in vivo (Figs. 6, 7, 8).