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C-myc-induced apoptosis in polycystic kidney disease is Bcl-2 and p53 independent.

Trudel M, Lanoix J, Barisoni L, Blouin MJ, Desforges M, L'Italien C, D'Agati V - J. Exp. Med. (1997)

Bottom Line: No renal abnormalities were detected in 13 transgenic lines established, indicating that the PKD phenotype is dependent on functions specific to c-myc.All SBM offspring, irrespective of their p53 genotype, developed PKD with increased renal epithelial apoptotic index.We conclude that the pathogenesis of PKD is c-myc specific and involves a critical imbalance between the opposing processes of cell proliferation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Faculté de Médecine de l'Université de Montréal, Montréal, Québec, Canada H2W 1R7.

ABSTRACT
The SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) induced by the dysregulated expression of c-myc in renal tissue. In situ hybridization analysis demonstrated intense signal for the c-myc transgene overlying tubular cystic epithelium in SBM mice. Renal proliferation index in SBM kidneys was 10-fold increased over nontransgenic controls correlating with the presence of epithelial hyperplasia. The specificity of c-myc for the proliferative potential of epithelial cells was demonstrated by substitution of c-myc with the proto-oncogene c-fos or the transforming growth factor (TGF)-alpha within the same construct. No renal abnormalities were detected in 13 transgenic lines established, indicating that the PKD phenotype is dependent on functions specific to c-myc. We also investigated another well characterized function of c-myc, the regulation of apoptosis through pathways involving p53 and members of the bcl-2 family, which induce and inhibit apoptosis, respectively. The SBM kidney tissues, which overexpress c-myc, displayed a markedly elevated (10-100-fold) apoptotic index. However, no significant difference in bcl-2, bax, or p53 expression was observed in SBM kidney compared with controls. Direct proof that the heightened renal cellular apoptosis in PKD is not occurring through p53 was obtained by successive matings between SBM and p53(-/-) mice. All SBM offspring, irrespective of their p53 genotype, developed PKD with increased renal epithelial apoptotic index. In addition, overexpression of both bcl-2 and c-myc in double transgenic mice (SBB+/SBM+) also produced a similar PKD phenotype with a high apoptotic rate, showing that c-myc can bypass bcl-2 in vivo. Thus, the in vivo c-myc apoptotic pathway in SBM mice occurs through a p53- and bcl-2-independent mechanism. We conclude that the pathogenesis of PKD is c-myc specific and involves a critical imbalance between the opposing processes of cell proliferation and apoptosis.

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p53-independent apoptosis in c-myc–induced PKD. (a) RT-PCR analysis of p53 expression (340-bp fragment) at different stages of development (0.5 d, 10 d, 4 mo, and end-stage renal disease, indicated above each lane) of a transgenic SBM line (SBM9) compared with control (negative  age-matched littermates). M, molecular weight marker; ESRD, end stage renal disease; d, day; m, month. (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1–4 μl) aliquots were amplified with primers specific for p53 and S16 as internal control. For semiquantitative evaluation, an acrylamide gel (bottom) was scanned by computerized densitometer and graphed (top). (Filled squares) p53, (open squares) S16. (c) Renal tissue of p53−/−SBM+  mice that shows numerous tubular cysts (hematoxylin-eosin, X30). (d) Apoptotic tubular epithelial cells are highlighted along the wall of a cyst and in the  cyst lumen of a SBM+p53−/− mouse (DAB chromogen, ×800). (e) p53−/−SBM+ renal tissue showing an apoptotic epithelial cell with nuclear fragmentation (arrow) being shed into the cyst lumen (hematoxylin-eosin, ×1,250).
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Figure 8: p53-independent apoptosis in c-myc–induced PKD. (a) RT-PCR analysis of p53 expression (340-bp fragment) at different stages of development (0.5 d, 10 d, 4 mo, and end-stage renal disease, indicated above each lane) of a transgenic SBM line (SBM9) compared with control (negative age-matched littermates). M, molecular weight marker; ESRD, end stage renal disease; d, day; m, month. (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1–4 μl) aliquots were amplified with primers specific for p53 and S16 as internal control. For semiquantitative evaluation, an acrylamide gel (bottom) was scanned by computerized densitometer and graphed (top). (Filled squares) p53, (open squares) S16. (c) Renal tissue of p53−/−SBM+ mice that shows numerous tubular cysts (hematoxylin-eosin, X30). (d) Apoptotic tubular epithelial cells are highlighted along the wall of a cyst and in the cyst lumen of a SBM+p53−/− mouse (DAB chromogen, ×800). (e) p53−/−SBM+ renal tissue showing an apoptotic epithelial cell with nuclear fragmentation (arrow) being shed into the cyst lumen (hematoxylin-eosin, ×1,250).

Mentions: p53 is a tumor suppressor gene that mediates c-myc-induced apoptosis (12, 38). Since c-myc is overexpressed in SBM kidney and renal tissues displayed a high apoptotic index, the level of p53 expression was investigated by RT-PCR analysis. Two transgenic mouse lines (SBM 9, SBM 75) at different stages of development (0.5 d, 10 d, 4 mo, end-stage) expressed approximately two- to threefold lower levels of p53 compared with negative age-matched littermates (Fig. 8 a), suggesting that p53 is not a major inducer of apoptosis in SBM. Proof that the expression analysis falls within the linear range is provided in Fig. 8 b.


C-myc-induced apoptosis in polycystic kidney disease is Bcl-2 and p53 independent.

Trudel M, Lanoix J, Barisoni L, Blouin MJ, Desforges M, L'Italien C, D'Agati V - J. Exp. Med. (1997)

p53-independent apoptosis in c-myc–induced PKD. (a) RT-PCR analysis of p53 expression (340-bp fragment) at different stages of development (0.5 d, 10 d, 4 mo, and end-stage renal disease, indicated above each lane) of a transgenic SBM line (SBM9) compared with control (negative  age-matched littermates). M, molecular weight marker; ESRD, end stage renal disease; d, day; m, month. (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1–4 μl) aliquots were amplified with primers specific for p53 and S16 as internal control. For semiquantitative evaluation, an acrylamide gel (bottom) was scanned by computerized densitometer and graphed (top). (Filled squares) p53, (open squares) S16. (c) Renal tissue of p53−/−SBM+  mice that shows numerous tubular cysts (hematoxylin-eosin, X30). (d) Apoptotic tubular epithelial cells are highlighted along the wall of a cyst and in the  cyst lumen of a SBM+p53−/− mouse (DAB chromogen, ×800). (e) p53−/−SBM+ renal tissue showing an apoptotic epithelial cell with nuclear fragmentation (arrow) being shed into the cyst lumen (hematoxylin-eosin, ×1,250).
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Related In: Results  -  Collection

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Figure 8: p53-independent apoptosis in c-myc–induced PKD. (a) RT-PCR analysis of p53 expression (340-bp fragment) at different stages of development (0.5 d, 10 d, 4 mo, and end-stage renal disease, indicated above each lane) of a transgenic SBM line (SBM9) compared with control (negative age-matched littermates). M, molecular weight marker; ESRD, end stage renal disease; d, day; m, month. (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1–4 μl) aliquots were amplified with primers specific for p53 and S16 as internal control. For semiquantitative evaluation, an acrylamide gel (bottom) was scanned by computerized densitometer and graphed (top). (Filled squares) p53, (open squares) S16. (c) Renal tissue of p53−/−SBM+ mice that shows numerous tubular cysts (hematoxylin-eosin, X30). (d) Apoptotic tubular epithelial cells are highlighted along the wall of a cyst and in the cyst lumen of a SBM+p53−/− mouse (DAB chromogen, ×800). (e) p53−/−SBM+ renal tissue showing an apoptotic epithelial cell with nuclear fragmentation (arrow) being shed into the cyst lumen (hematoxylin-eosin, ×1,250).
Mentions: p53 is a tumor suppressor gene that mediates c-myc-induced apoptosis (12, 38). Since c-myc is overexpressed in SBM kidney and renal tissues displayed a high apoptotic index, the level of p53 expression was investigated by RT-PCR analysis. Two transgenic mouse lines (SBM 9, SBM 75) at different stages of development (0.5 d, 10 d, 4 mo, end-stage) expressed approximately two- to threefold lower levels of p53 compared with negative age-matched littermates (Fig. 8 a), suggesting that p53 is not a major inducer of apoptosis in SBM. Proof that the expression analysis falls within the linear range is provided in Fig. 8 b.

Bottom Line: No renal abnormalities were detected in 13 transgenic lines established, indicating that the PKD phenotype is dependent on functions specific to c-myc.All SBM offspring, irrespective of their p53 genotype, developed PKD with increased renal epithelial apoptotic index.We conclude that the pathogenesis of PKD is c-myc specific and involves a critical imbalance between the opposing processes of cell proliferation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Faculté de Médecine de l'Université de Montréal, Montréal, Québec, Canada H2W 1R7.

ABSTRACT
The SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) induced by the dysregulated expression of c-myc in renal tissue. In situ hybridization analysis demonstrated intense signal for the c-myc transgene overlying tubular cystic epithelium in SBM mice. Renal proliferation index in SBM kidneys was 10-fold increased over nontransgenic controls correlating with the presence of epithelial hyperplasia. The specificity of c-myc for the proliferative potential of epithelial cells was demonstrated by substitution of c-myc with the proto-oncogene c-fos or the transforming growth factor (TGF)-alpha within the same construct. No renal abnormalities were detected in 13 transgenic lines established, indicating that the PKD phenotype is dependent on functions specific to c-myc. We also investigated another well characterized function of c-myc, the regulation of apoptosis through pathways involving p53 and members of the bcl-2 family, which induce and inhibit apoptosis, respectively. The SBM kidney tissues, which overexpress c-myc, displayed a markedly elevated (10-100-fold) apoptotic index. However, no significant difference in bcl-2, bax, or p53 expression was observed in SBM kidney compared with controls. Direct proof that the heightened renal cellular apoptosis in PKD is not occurring through p53 was obtained by successive matings between SBM and p53(-/-) mice. All SBM offspring, irrespective of their p53 genotype, developed PKD with increased renal epithelial apoptotic index. In addition, overexpression of both bcl-2 and c-myc in double transgenic mice (SBB+/SBM+) also produced a similar PKD phenotype with a high apoptotic rate, showing that c-myc can bypass bcl-2 in vivo. Thus, the in vivo c-myc apoptotic pathway in SBM mice occurs through a p53- and bcl-2-independent mechanism. We conclude that the pathogenesis of PKD is c-myc specific and involves a critical imbalance between the opposing processes of cell proliferation and apoptosis.

Show MeSH
Related in: MedlinePlus