Table 1.
Origin and characteristics of human cell line models under investigation.
Figure 1.
Protein and activity levels of BCR-ABL and Separase in cell lines under investigation.
Protein levels of p210BCR-ABL (A) and Separase (C) based on densitometric evaluation of immunostained Western blots were normalized to Actin as loading control. Abl-related TK activity (BCR-ABL+c-ABL) was measured as pCrkL/Actin (B). Separase proteolytic activity (D) was quantified using an in vitro fluorometric assay and was given as relative fluorescence units/Actin (RFU/Actin). Analyses were performed on protein lysates derived from p210BCR-ABL-positive (LAMA-84, K562) and -negative cells (NHDF, UROtsa, HL-60, U937), and from induced U937p210BCR-ABL/c6 cells (U937/c6-On) expressing a p210BCR-ABL transgene under control of a Doxycycline inducible promoter.
Figure 2.
Transcript levels, protein levels and proteolytic activity of Separase in BCR-ABL-negative cells treated with IM.
Cells were treated individually with distinct concentrations (0.5 to 10 µM) of IM. After about two cell cycle rounds (NHDF, UROtsa, 6 d; HL-60 and U937, 48 h) total RNA and protein lysates were prepared and analyzed by qRT-PCR (A), Western blot immunostaining (B) and Separase fluorometric activity assays (C). For Westren blot experiments, Actin served as loading control and/or for densitometric data normalization. Each data point corresponds to one single experiment. Only significant p-values as calculated between treated and untreated cells were shown (see Table 2 for summarized Δ-values). For a representative set of corresponding immunostained Western blots compare Figure 5 panel B.
Table 2.
Percent changes (Δ-values) in transcript levels, protein levels and proteolytic activity of Separase after IM treatment when compared to the corresponding untreated cells.1, 2
Figure 3.
Cell cycle profiles of untreated and IM-treated cell lines.
Triplicate cell cultures were incubated with IM at times and doses where significant changes in Separase protein levels and/or Separase proteolytic activation have been observed (Figure 2 and 4). Subsequently, the cell cycle profile was analyzed by flow cytometry after propidium iodide staining. The percentages of cells in G2/M, S, G1/G0 phases and in apoptotic state are depicted in each bar.
Figure 4.
Transcript levels, protein levels and proteolytic activity of Separase in BCR-ABL-positive cells treated with IM.
Cells were treated individually with distinct concentrations (0.5 to 5 µM) of IM. After about two cell cycle rounds (K562, LAMA-84, 24 h; U937p210BCR-ABL/c6-On, 48 h) total RNA and protein lysates were prepared and analyzed by qRT-PCR (A), Western blot immunostaining (B) and separase fluorometric activity assays (C). For Western blot experiments, Actin served as loading control and/or for densitometric data normalization. Each data point corresponds to one single experiment. Only significant p-values as calculated between treated and untreated cells were shown (see Table 2 for summarized Δ-values). For a representative set of corresponding immunostained Western blots compare Figure 5 panel C. Abbreviations: U937/c6-On, U937 cells expressing a p210BCR-ABL transgene (Tet-On system) after induction with Doxycycline (U937p210BCR-ABL/c6-On).
Figure 5.
Analysis of master Separase proteolytic activity regulators in BCR-ABL-negative and -positive cell lines treated with IM.
Schematic diagram of cooperating inhibitory factors that regulate Separase proteolytic activity in a tight cell cycle controlled manner (A). Degradation of Securin, inactivation of the CyclinB1/CDK1 complex, dephosphorylation of Separase at a specific serine residue (pSer1126) by the anaphase promoting complex/cyclosome (APC/C), and the release of PP2A contribute to activation of Separase. Representative composite image of Western blot immunostaining experiments illustrate the expression levels of Separase and relevant regulatory proteins (Securin, pSer1126, CyclinB1 and PP2A) in BCR-ABL-negative (B) and BCR-ABL-positive (C) cell lines. Images are cropped sections derived from stripped and reprobed Western blot immunostainings used for acquisition of densitometric data shown in Figures 2 and 4. Cells were treated with IM for times and doses given on top. Actin served as loading control. The densitometric data of at least triplicate experiments are presented in Table 3.
Table 3.
Percent changes (Δ-values) in regulator levels of Separase proteolytic activity after IM treatment when compared to the corresponding untreated cells.1