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Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation.

Scott CL, Schuler M, Marsden VS, Egle A, Pellegrini M, Nesic D, Gerondakis S, Nutt SL, Green DR, Strasser A - J. Cell Biol. (2004)

Bottom Line: Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors.Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas.Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.

ABSTRACT
Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1(-/-) and caspase-9(-/-) mice. Due to perinatal lethality, Emu-myc transgenic Apaf-1(-/-) or caspase-9(-/-) fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.

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Sensitivity of preneoplastic B lymphoid cells to apoptotic stimuli. Pre–B cells (B220+sIg−) and B cells (B220+sIg+) were purified from bone marrow and lymph nodes, respectively, of recipient mice showing no signs of lymphoma by immunofluorescent staining and sorting on a FACStar, DIVA, or MoFlo. (A–D) Sensitivity of preneoplastic B cells to the following apoptotic stimuli: culture in the absence of cytokines (A), 100 nM dexamethasone (B), 10 μg/ml etoposide (VP16; C), and 10 Gy γ-irradiation (D). Eμ-myc (open circle), Eμ-myc/Apaf-1−/− (closed circle), Apaf-1+/+ (open square), and Apaf-1−/− (closed square). Cells were cultured and viability was determined over 6–72 h as described in Fig. 4 legend. (E) The colony-forming potential of pro/pre–B cells from the bone marrow of C57BL/6-Ly5.1 mice reconstituted with Eμ-myc or Eμ-myc/Apaf-1−/− fetal liver stem cells in limiting dilution after no treatment or after exposure to 2.5 Gy γ-radiation or 1.0, 0.3, 0.03 μg/ml etoposide (VP16). B cell colonies consisting of at least 20 cells were scored blinded as to genotype using an inverted microscope at day 10, and the cloning frequency was determined using limiting dilution analysis. Clonogenicity is less than or equal to the value shown. Data represent arithmetic means ± SD of 3–10 mice of each genotype.
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fig6: Sensitivity of preneoplastic B lymphoid cells to apoptotic stimuli. Pre–B cells (B220+sIg−) and B cells (B220+sIg+) were purified from bone marrow and lymph nodes, respectively, of recipient mice showing no signs of lymphoma by immunofluorescent staining and sorting on a FACStar, DIVA, or MoFlo. (A–D) Sensitivity of preneoplastic B cells to the following apoptotic stimuli: culture in the absence of cytokines (A), 100 nM dexamethasone (B), 10 μg/ml etoposide (VP16; C), and 10 Gy γ-irradiation (D). Eμ-myc (open circle), Eμ-myc/Apaf-1−/− (closed circle), Apaf-1+/+ (open square), and Apaf-1−/− (closed square). Cells were cultured and viability was determined over 6–72 h as described in Fig. 4 legend. (E) The colony-forming potential of pro/pre–B cells from the bone marrow of C57BL/6-Ly5.1 mice reconstituted with Eμ-myc or Eμ-myc/Apaf-1−/− fetal liver stem cells in limiting dilution after no treatment or after exposure to 2.5 Gy γ-radiation or 1.0, 0.3, 0.03 μg/ml etoposide (VP16). B cell colonies consisting of at least 20 cells were scored blinded as to genotype using an inverted microscope at day 10, and the cloning frequency was determined using limiting dilution analysis. Clonogenicity is less than or equal to the value shown. Data represent arithmetic means ± SD of 3–10 mice of each genotype.

Mentions: Deregulated c-myc expression increases susceptibility of lymphocytes and many other cell types to a broad range of apoptotic stimuli (Pelengaris et al., 2002). Therefore, we FACS®-sorted preneoplastic Eμ-myc/Apaf-1+/+ and Eμ-myc/Apaf-1−/− pre–B cells and B cells from reconstituted animals and investigated whether loss of Apaf-1 rendered them resistant to cytokine withdrawal, dexamethasone, etoposide, or γ-radiation in culture. A modest (<1.5-fold) reduction in sensitivity to these stress stimuli was observed for pre–B cells at 6, 24, and 48 h (P < 0.05 at 24 h, t test); however, by 72 h, no consistent difference in cell death was apparent with loss of Apaf-1 (Fig. 6, A–D). These conclusions were based on short-term survival assays. A more stringent measurement of cell survival is to determine whether cells retain colony forming potential. As pro/pre–B cells can be grown as colonies on stromal cells in the presence of IL-7 (Rolink et al., 1991), we investigated whether loss of Apaf-1 had an effect on clonogenic survival of Eμ-myc pro/pre–B cells. Eμ-myc/Apaf-1+/+ pro/pre–B cells formed colonies at a frequency of 1/14 ± 12 (Fig. 6 E) and loss of Apaf-1 did not alter this (Eμ-myc/Apaf-1−/− 1/12 ± 8; P = 0.5). When colony forming cells were exposed to γ-radiation (2.5 Gy) or a 6-h treatment with etoposide (0.3–1.0 μg/ml), the clonal frequency decreased by 10–80-fold, and loss of Apaf-1 did not afford any protection (P = 0.6; Fig. 6 E). Thus, the protection against apoptosis seen at early time points (24–48 h) in short-term survival assays could be compensated for in the longer term by other apoptotic regulators. Collectively, these results demonstrate that Apaf-1 plays no, or only a minor, role in cell death that is enhanced by deregulated c-myc expression.


Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation.

Scott CL, Schuler M, Marsden VS, Egle A, Pellegrini M, Nesic D, Gerondakis S, Nutt SL, Green DR, Strasser A - J. Cell Biol. (2004)

Sensitivity of preneoplastic B lymphoid cells to apoptotic stimuli. Pre–B cells (B220+sIg−) and B cells (B220+sIg+) were purified from bone marrow and lymph nodes, respectively, of recipient mice showing no signs of lymphoma by immunofluorescent staining and sorting on a FACStar, DIVA, or MoFlo. (A–D) Sensitivity of preneoplastic B cells to the following apoptotic stimuli: culture in the absence of cytokines (A), 100 nM dexamethasone (B), 10 μg/ml etoposide (VP16; C), and 10 Gy γ-irradiation (D). Eμ-myc (open circle), Eμ-myc/Apaf-1−/− (closed circle), Apaf-1+/+ (open square), and Apaf-1−/− (closed square). Cells were cultured and viability was determined over 6–72 h as described in Fig. 4 legend. (E) The colony-forming potential of pro/pre–B cells from the bone marrow of C57BL/6-Ly5.1 mice reconstituted with Eμ-myc or Eμ-myc/Apaf-1−/− fetal liver stem cells in limiting dilution after no treatment or after exposure to 2.5 Gy γ-radiation or 1.0, 0.3, 0.03 μg/ml etoposide (VP16). B cell colonies consisting of at least 20 cells were scored blinded as to genotype using an inverted microscope at day 10, and the cloning frequency was determined using limiting dilution analysis. Clonogenicity is less than or equal to the value shown. Data represent arithmetic means ± SD of 3–10 mice of each genotype.
© Copyright Policy
Related In: Results  -  Collection

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fig6: Sensitivity of preneoplastic B lymphoid cells to apoptotic stimuli. Pre–B cells (B220+sIg−) and B cells (B220+sIg+) were purified from bone marrow and lymph nodes, respectively, of recipient mice showing no signs of lymphoma by immunofluorescent staining and sorting on a FACStar, DIVA, or MoFlo. (A–D) Sensitivity of preneoplastic B cells to the following apoptotic stimuli: culture in the absence of cytokines (A), 100 nM dexamethasone (B), 10 μg/ml etoposide (VP16; C), and 10 Gy γ-irradiation (D). Eμ-myc (open circle), Eμ-myc/Apaf-1−/− (closed circle), Apaf-1+/+ (open square), and Apaf-1−/− (closed square). Cells were cultured and viability was determined over 6–72 h as described in Fig. 4 legend. (E) The colony-forming potential of pro/pre–B cells from the bone marrow of C57BL/6-Ly5.1 mice reconstituted with Eμ-myc or Eμ-myc/Apaf-1−/− fetal liver stem cells in limiting dilution after no treatment or after exposure to 2.5 Gy γ-radiation or 1.0, 0.3, 0.03 μg/ml etoposide (VP16). B cell colonies consisting of at least 20 cells were scored blinded as to genotype using an inverted microscope at day 10, and the cloning frequency was determined using limiting dilution analysis. Clonogenicity is less than or equal to the value shown. Data represent arithmetic means ± SD of 3–10 mice of each genotype.
Mentions: Deregulated c-myc expression increases susceptibility of lymphocytes and many other cell types to a broad range of apoptotic stimuli (Pelengaris et al., 2002). Therefore, we FACS®-sorted preneoplastic Eμ-myc/Apaf-1+/+ and Eμ-myc/Apaf-1−/− pre–B cells and B cells from reconstituted animals and investigated whether loss of Apaf-1 rendered them resistant to cytokine withdrawal, dexamethasone, etoposide, or γ-radiation in culture. A modest (<1.5-fold) reduction in sensitivity to these stress stimuli was observed for pre–B cells at 6, 24, and 48 h (P < 0.05 at 24 h, t test); however, by 72 h, no consistent difference in cell death was apparent with loss of Apaf-1 (Fig. 6, A–D). These conclusions were based on short-term survival assays. A more stringent measurement of cell survival is to determine whether cells retain colony forming potential. As pro/pre–B cells can be grown as colonies on stromal cells in the presence of IL-7 (Rolink et al., 1991), we investigated whether loss of Apaf-1 had an effect on clonogenic survival of Eμ-myc pro/pre–B cells. Eμ-myc/Apaf-1+/+ pro/pre–B cells formed colonies at a frequency of 1/14 ± 12 (Fig. 6 E) and loss of Apaf-1 did not alter this (Eμ-myc/Apaf-1−/− 1/12 ± 8; P = 0.5). When colony forming cells were exposed to γ-radiation (2.5 Gy) or a 6-h treatment with etoposide (0.3–1.0 μg/ml), the clonal frequency decreased by 10–80-fold, and loss of Apaf-1 did not afford any protection (P = 0.6; Fig. 6 E). Thus, the protection against apoptosis seen at early time points (24–48 h) in short-term survival assays could be compensated for in the longer term by other apoptotic regulators. Collectively, these results demonstrate that Apaf-1 plays no, or only a minor, role in cell death that is enhanced by deregulated c-myc expression.

Bottom Line: Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors.Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas.Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.

ABSTRACT
Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1(-/-) and caspase-9(-/-) mice. Due to perinatal lethality, Emu-myc transgenic Apaf-1(-/-) or caspase-9(-/-) fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.

Show MeSH
Related in: MedlinePlus