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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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Loss of Apaf-1 protects MEFs against rapid p53-dependent apoptosis, but not against p53-dependent suppression of long-term proliferative survival. (A) Early passage MEFs of the indicated genotypes were retrovirally transduced to express E1A plus mutant ras or E1A, mutant ras, and Bcl-xL. Apoptotic cell death was assessed by staining with FITC-coupled Annexin V plus PI and flow cytometry at the indicated time points after serum withdrawal or 24 h after UV radiation, and the mean fractions of Annexin V−/PI− viable cells are given. (B) Early passage MEFs of the indicated genotypes were retrovirally transduced to express the indicated combinations of oncogenes. 2 d after transduction, the cells were seeded in triplicate at low density in selection medium. After 7 d culture in selection medium, cells were fixed, stained, and, the resulting colonies, enumerated. Mean value ± SD of three independent experiments.
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fig7: Loss of Apaf-1 protects MEFs against rapid p53-dependent apoptosis, but not against p53-dependent suppression of long-term proliferative survival. (A) Early passage MEFs of the indicated genotypes were retrovirally transduced to express E1A plus mutant ras or E1A, mutant ras, and Bcl-xL. Apoptotic cell death was assessed by staining with FITC-coupled Annexin V plus PI and flow cytometry at the indicated time points after serum withdrawal or 24 h after UV radiation, and the mean fractions of Annexin V−/PI− viable cells are given. (B) Early passage MEFs of the indicated genotypes were retrovirally transduced to express the indicated combinations of oncogenes. 2 d after transduction, the cells were seeded in triplicate at low density in selection medium. After 7 d culture in selection medium, cells were fixed, stained, and, the resulting colonies, enumerated. Mean value ± SD of three independent experiments.

Mentions: As aforementioned, the hypothesis that Apaf-1 and caspase-9 function as tumor suppressor genes was based on the observation that the loss of these proteins increased colony formation of MEFs infected with viruses encoding the oncogenes myc plus or minus mutant ras (Soengas et al., 1999). We performed similar experiments with the adenoviral oncogene E1A or c-myc, plus or minus mutant ras, but found no increase in oncogene-induced colony formation of early passage MEFs with loss of Apaf-1 (Fig. 7). In short-term assays, MEFs lacking Apaf-1, Bax, or p53 or expressing Bcl-xL were resistant to apoptosis induction in response to serum withdrawal or UV-C (Fig. 7 A), and similar results were obtained with etoposide, actinomycin D, staurosporine, doxorubicine, cisplatin, and taxol (not depicted). However, in longer-term studies requiring cellular transformation, loss of Apaf-1 did not result in a growth advantage (Fig. 7 B). In contrast, a large increase in colony formation was observed when MEFs from p53 or Bax-deficient mice were infected with E1A plus mutant ras- or c-myc plus mutant ras-containing viruses, but not when Apaf-1−/− MEFs were used (Fig. 7 B). All MEFs grew at comparable rates under standard cell culture conditions (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200310041/DC1), and the oncogenic proteins were expressed at similar levels in each of the MEFs infected with oncogene-containing retroviruses (Fig. S2 B). Comparable results were obtained in three independent experiments performed with separately derived sets of MEFs of each genotype. In addition, the inability of the Apaf-1 deficiency to enhance transformation in focus assays was observed in MEFs from two independently generated knockout mouse lines (Cecconi et al., 1998; Yoshida et al., 1998; unpublished data). The ability of p53 and Bax to suppress cell transformation in this system is consistent with observations that induction of apoptosis is critical for p53's tumor suppressive effects (Schmitt et al., 2002) and that such apoptosis can be activated via Bax (McCurrach et al., 1997; Zhang et al., 2000). Therefore, under the conditions we used, a block to apoptosis upstream of the mitochondria can promote transformation, which is not seen when the defect is at the level of Apaf-1.


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)

Loss of Apaf-1 protects MEFs against rapid p53-dependent apoptosis, but not against p53-dependent suppression of long-term proliferative survival. (A) Early passage MEFs of the indicated genotypes were retrovirally transduced to express E1A plus mutant ras or E1A, mutant ras, and Bcl-xL. Apoptotic cell death was assessed by staining with FITC-coupled Annexin V plus PI and flow cytometry at the indicated time points after serum withdrawal or 24 h after UV radiation, and the mean fractions of Annexin V−/PI− viable cells are given. (B) Early passage MEFs of the indicated genotypes were retrovirally transduced to express the indicated combinations of oncogenes. 2 d after transduction, the cells were seeded in triplicate at low density in selection medium. After 7 d culture in selection medium, cells were fixed, stained, and, the resulting colonies, enumerated. Mean value ± SD of three independent experiments.
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Related In: Results  -  Collection

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fig7: Loss of Apaf-1 protects MEFs against rapid p53-dependent apoptosis, but not against p53-dependent suppression of long-term proliferative survival. (A) Early passage MEFs of the indicated genotypes were retrovirally transduced to express E1A plus mutant ras or E1A, mutant ras, and Bcl-xL. Apoptotic cell death was assessed by staining with FITC-coupled Annexin V plus PI and flow cytometry at the indicated time points after serum withdrawal or 24 h after UV radiation, and the mean fractions of Annexin V−/PI− viable cells are given. (B) Early passage MEFs of the indicated genotypes were retrovirally transduced to express the indicated combinations of oncogenes. 2 d after transduction, the cells were seeded in triplicate at low density in selection medium. After 7 d culture in selection medium, cells were fixed, stained, and, the resulting colonies, enumerated. Mean value ± SD of three independent experiments.
Mentions: As aforementioned, the hypothesis that Apaf-1 and caspase-9 function as tumor suppressor genes was based on the observation that the loss of these proteins increased colony formation of MEFs infected with viruses encoding the oncogenes myc plus or minus mutant ras (Soengas et al., 1999). We performed similar experiments with the adenoviral oncogene E1A or c-myc, plus or minus mutant ras, but found no increase in oncogene-induced colony formation of early passage MEFs with loss of Apaf-1 (Fig. 7). In short-term assays, MEFs lacking Apaf-1, Bax, or p53 or expressing Bcl-xL were resistant to apoptosis induction in response to serum withdrawal or UV-C (Fig. 7 A), and similar results were obtained with etoposide, actinomycin D, staurosporine, doxorubicine, cisplatin, and taxol (not depicted). However, in longer-term studies requiring cellular transformation, loss of Apaf-1 did not result in a growth advantage (Fig. 7 B). In contrast, a large increase in colony formation was observed when MEFs from p53 or Bax-deficient mice were infected with E1A plus mutant ras- or c-myc plus mutant ras-containing viruses, but not when Apaf-1−/− MEFs were used (Fig. 7 B). All MEFs grew at comparable rates under standard cell culture conditions (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200310041/DC1), and the oncogenic proteins were expressed at similar levels in each of the MEFs infected with oncogene-containing retroviruses (Fig. S2 B). Comparable results were obtained in three independent experiments performed with separately derived sets of MEFs of each genotype. In addition, the inability of the Apaf-1 deficiency to enhance transformation in focus assays was observed in MEFs from two independently generated knockout mouse lines (Cecconi et al., 1998; Yoshida et al., 1998; unpublished data). The ability of p53 and Bax to suppress cell transformation in this system is consistent with observations that induction of apoptosis is critical for p53's tumor suppressive effects (Schmitt et al., 2002) and that such apoptosis can be activated via Bax (McCurrach et al., 1997; Zhang et al., 2000). Therefore, under the conditions we used, a block to apoptosis upstream of the mitochondria can promote transformation, which is not seen when the defect is at the level of Apaf-1.

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