The following rabbit monoclonal antibodies were used: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E, Cell Signaling Technology), and p21 Waf1/Cip1 (12D1, Cell Signaling Technology). abnormalities, but not continuous stimulation of growth signaling, possibly through stochastic genetic alterations. Introduction Src-family non-receptor-type tyrosine kinases play a key role in the regulation of diverse cellular functions, including cell proliferation, differentiation, adhesion, and motility1C3. c-Src, a member of the Src-family, is usually ubiquitously expressed in various types of tissues and cells. LY-2584702 c-Src is usually overexpressed and abnormally activated in cancer cells, and the kinase activity of c-Src is usually correlated with progression of human cancers4,5. Although the activity of c-Src is usually tightly regulated in normal cells, deregulation of its activity often causes tumorigenesis. In particular, v-Src is the first identified oncogene product isolated from Rous sarcoma LY-2584702 computer virus, and the kinase activity of v-Src is usually drastically increased compared with that of c-Src due to the presence of several point mutations and the lack of the C-terminal unfavorable regulatory region1,6C8. Colony formation assays have shown that expression of v-Src causes anchorage-independent and infinite cell proliferation. All of v-Src-expressing cells are supposed to form transformed colonies. However, abnormally LY-2584702 low frequencies of the formation of v-Src-induced transformed colonies have been indicated in the literature9C11. These low frequencies of colony formation would be inconsistent with common efficiencies of v-Src contamination or Nfia transfection (approximately 5~80%). In this study, to clarify a quantitative relationship between v-Src expression and colony formation, we utilized cell lines expressing tetracycline-inducible v-Src. Despite accomplishment of extremely high expression efficiencies of v-Src (nearly 100%), the frequencies of v-Src-induced colony formation were as low as those indicated in the literature. We revealed that inducible expression of LY-2584702 v-Src up-regulates the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest, even though the ERK/MAPK pathway was simultaneously activated. We further showed that inducible expression of v-Src brings about chromosome abnormalities and approximately half of v-Src-expressing cells subsequently suppress v-Src expression. More importantly, a limited number of v-Src-suppressed cells can only start to re-proliferate vigorously and form transformed colonies. We thus provide evidence for a surprising role of v-Src in cell transformation. Results Cell cycle arrest by v-Src-induced tyrosine phosphorylation in HeLa S3 cells In our recent study, we generated HeLa S3 and HCT116 cells, both of which express tetracycline-inducible wild-type v-Src (HeLa S3/TR/v-Src-wt and HCT116/TR/v-Src-wt), and showed that inducible expression of v-Src-wt forces the cells to become rounded and detached from a culture dish, which surprisingly leads to inhibition of cell proliferation in a manner dependent on the expression level of v-Src-wt12. To examine the expression efficiency of v-Src-wt in HeLa S3/TR/v-Src-wt cells, we immunostained HeLa S3/TR/v-Src-wt cells with anti-Src (#327) antibody. Confocal microscopic and flow cytometric analyses showed the extremely high efficiency of v-Src-wt expression upon addition of doxycycline (Dox), a tetracycline derivative (Supplementary Fig.?1a,1b). Western blotting analysis confirmed Dox-dependent expression of v-Src-wt using anti-Src (N-16) antibody, which preferentially recognizes v-Src compared with c-Src (Supplementary Fig.?1c). Furthermore, using anti-Src (#327) antibody, which is usually capable of recognizing both c-Src and v-Src, we showed LY-2584702 that the level of induced v-Src-wt expression was lower than that of endogenous c-Src. However, the level of protein tyrosine phosphorylation was dramatically increased by v-Src-wt expression due to the point mutations and the lack of the C-terminal unfavorable regulatory region (Supplementary Fig.?1c). From 2?h after Dox addition, expression of v-Src-wt and tyrosine phosphorylation of cellular proteins were appreciably increased in a time-dependent manner (Supplementary Fig.?1d). These results suggest that HeLa S3/TR/v-Src-wt cells exhibit the extremely high efficiency of v-Src expression upon Dox treatment. To examine the effect of v-Src on cell cycle progression, HeLa S3/TR/v-Src-wt cells were highly synchronized at the G1/S boundary by a double thymidine block (Fig.?1a). After release from the G1/S boundary, expression of v-Src-wt was induced by Dox treatment and DNA contents were analyzed by flow cytometry. v-Src-wt-expressing cells appeared to normally progress into G2/M phase 12?h after release from a double thymidine block (Fig.?1b). However, 24?h after the release, v-Src-wt expression inhibited cell cycle progression largely in G1 phase and slightly in G2 phase (Fig.?1b). To further examine whether the kinase activity of v-Src was indispensable to v-Src-mediated cell-cycle arrest, we generated.