Construction and use of super module vector for the induction of c-SrcY527F in human colon cancer cells. (A) Schematic illustrating the construction and structural features of the pSMVLuc-Src527F vector. The plasmid map of pSMVLuc-Src527F shows component elements of the vector, including a β-actin promoter-driven EGFP-linked module expressing rtTA2S-M2 transcriptional transactivator, a kanamycin/neomycin resistance cassette, and a bidirectional promoter-based module expressing luciferase and c-SrcY527F in a Dox-dependent manner. (B) Comparison of endogenous c-Src levels in HT29, WiDr, HCT116, and SW480 colon cancer cell lines. Int 407 cells represent a nontransformed human intestinal epithelial cell line [37]. (C) c-SrcY527F induction in representative HCT116 Luc-SrcY527F clones determined after 24 hours in the presence of Dox. (D) Growth characteristics of parental wt HCT116 cells and selected HCT116 Luc-only, Luc-wtSrc, and Luc-SrcY527F clones in the absence of Dox. Growth curves were determined after seeding 1 x 10⁵ cells/well in six-well plates. Data are representative of three independent experiments performed in triplicate. Error bars represent standard deviations. For better transparency, the growth curves for each clone are also presented individually in Figure W4. Figure 1 contains a supplementary panel (Figure W1) illustrating the phosphorylation of diverse downstream effectors of c-Src in selected HCT116 clones in the presence and in the absence of Dox.

Consequences of the high-level induction of c-SrcY527F in HCT116 colorectal cancer cells. (A) Representative pictures of HCT116 Luc-Src527F High (clone HighY50) and control HCT116 Luc-only (clone LucC6) cells 24 hours after induction. Fluorescent microscopy insert illustrates lack of EGFP signals in vesicles formed after a high-level expression of c-SrcY527F. (B) Western blot analyses showing increases in the expression level of total and autophosphorylated c-Src [Tyr416] 24 hours after induction in HCT116 Luc-Src527F High clones but not in HCT116 Luc-only controls and wt HCT116 cells. (C) Quantification of adherent and detached cells in indicated Dox-induced and uninduced HCT116 Luc-Src527F High clones, HCT116 Luc-only control cell lines, and parental HCT116 wt cells. Cell counts were performed 24 hours after induction, and data are based on three independent experiments performed in triplicate. All HCT116 Luc-Src527F High clones displayed significantly decreased total cell numbers and increased numbers of detached cells following induction with Dox (two-sided t-tests, P < .01). A slight decrease in total cell number following Dox treatment was also observed for clone HCT116 Luc-only C6 (P < .05), but this was caused by a mechanism different from that observed for HCT116 Luc-Src527F High clones (Figure 4). (D) Four-day growth curves of HCT116 Luc-Src527F HighY50 cells, and HCT116 Luc-only, and LucB20 controls grown in the absence and in the presence of Dox. The initial seeding density was 4 x 10⁵ cells/well. Data are representative of three independent experiments performed in triplicate.

Increased levels and activity of Src do not promote the growth of the fraction of HCT116 Luc-Src527F cells that overcame the growth delay induced by a high-level expression of c-SrcY527F. (A) Schematic illustrating the generation and morphologic properties of “post-growth-delay” cells. Western blot analyses show the levels of total and autophosphorylated [Tyr416] Src in “post-growth-delay” cells and matched controls grown for 6 days in the presence or in the absence of Dox (end point of experiments presented in B). (B) Growth properties of “post-growth-delay” HCT116 Luc-Src527F High cells (clones HighX45 and HighY50) maintained for 6 days in the presence or in the absence of Dox. Identically treated HCT116 Luc-only cells (clone LucB20) and wt HCT116 cells were used as controls. The initial seeding density was 1 x 10⁵ cells/well in a six-well plate, and the medium was replaced by fresh medium on day 3. For each day, data are presented as the ratio of total cell number in the presence of Dox to the total number of the same cells in the absence of Dox, and are normalized to the results obtained with wt HCT116 cells. Normalization was performed to compensate for unspecific inhibitory effects of prolonged Dox treatment. Data are representative of three independent experiments performed in triplicate.

Consequences of the induction of wt c-Src (A) and c-SrcY527F (B) on the growth of HCT116 cells in a xenograft model. There were at least five animals in each control group and Dox-treated group. Successful gene induction in Dox-treated animals was confirmed using luciferase reporter assay (histogram insert). The use of a Dox-containing diet did not affect animal body weight during the experiment (line-plot insert).

Consequences of the overexpression of wt c-Src and the low-level induction of c-SrcY527F in HCT116 colorectal cancer cells. (A) Western blot analyses illustrating the levels of total and autophosphorylated active Src [Tyr416] in indicated clones 24 hours after induction. (B) Histogram showing no changes in cell proliferation 24 hours following high-level induction of wt c-Src (clones wt23 and wt28) and low-level induction of c-SrcY527F (clones LowX43 and LowY12). Data are based on three independent experiments performed in triplicate. No statistically significant differences were found between numbers of cells in the presence and in the absence of Dox for any of the clones (two-sided t-tests, P > .05). (C) Four-day growth curves of HCT116 Luc-wtSrc (clone wt28) and HCT116 Luc-Src527F (clone LowX43) cells grown in the absence and in the presence of Dox. The initial seeding density was 4 x 10⁵ cells/well. Data are representative of three independent experiments performed in triplicate. The cell cycle profiles of HCT116 Luc-wtSrc cells and HCT Luc-Src527F Low cells are presented in Figure W2.

Effects of a high-level induction of c-SrcY527F on cell cycle profile and cell viability in HCT116 colorectal carcinoma cells. (A) Cell cycle profiles of HCT116 Luc-only cells (clone LucB20) and HCT116 Luc-Src527F cells (clone HighY50) obtained after 24 hours of growth in the presence or in the absence of Dox. Data are representative of three independent experiments. Results obtained with other Luc-only and Luc-Src527F High clones analyzed were similar to those using clones LucB20 and Luc-Src527F HighY50, respectively. (B) Histogram illustrating relative increases in the percentage of cells in the G₂/M phase of the cell cycle after 24 hours of growth in the presence of Dox in HCT116 Luc-Src527F High cells but not in control HCT116 Luc-only clones. Data are based on three independently obtained cell cycle profiles. Western blot analyses show an accompanying decrease in levels of phosphorylated histone H3 [P-Ser10] and an increase in the phosphorylation of Cdk1 [P-Tyr15], confirming the accumulation of cells in the G₂ phase rather than in the M phase of the cell cycle [38]. They are representative of three repeat experiments. (C) Increases in the percentage of dead cells 24 and 48 hours after induction with Dox in HCT116 Luc-Src527F High cells but not in control clones. Determined using trypan blue exclusion method. Representative of three independent experiments performed in triplicate. (D) Histogram illustrating the levels of apoptosis-associated M30 antigen in the conditioned medium of indicated HCT116 Luc-Src527F High clones, control Luc-only cell lines, and parental wt HCT116 cells grown for 24 hours in the absence or in the presence of Dox. Staurosporine (35 nM)-treated cells represent positive control for ∼50% apoptotic cells. Data are representative of three independent experiments performed in triplicate. Lack of apoptotic phenotype following a high-level induction of c-SrcY527F was also independently confirmed by DAPI staining (Figure W3). (E) Representative electron microscopy pictures illustrating ultrastructural changes occurring in HCT116 cells following a high-level induction of c-SrcY527F.

Consequences of low-level and high-level induction of c-SrcY527F in SW480 colorectal cancer cells. Histogram shows cell numbers in induced and uninduced SW480 Luc-Src527F Low, SW480 Luc-Src527F High, and parental SW480 cells 24 hours after Dox treatment. Data are based on three independent experiments performed in triplicate. SW480 Luc-SrcY527F High cells displayed a significantly decreased total cell number following induction with Dox compared to noninduced cells (two-sided t-test, P < .05). No statistically significant differences were found between the numbers of wt SW480 and SW480 Luc-Src527F Low cells in the presence and in the absence of Dox (P > .05). Western blot analysis illustrates increases in the amount of Src following Dox induction.