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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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Expression analysis  of the SBF transgenic mice. (a)  RT-PCR expression analysis of  SBF (234-bp fragment) in 4 different transgenic lines (SBF 38,  19, 29, and 20) shows strong expression of the SBF construct in  kidney (K) and spleen (S), but not  in liver (Li), heart (H), lung (Lu),  or brain (B), shown for SBF 38  only). SBF38 shows high expression of the transgene in kidney  and spleen and weak expression  in lung and brain. M, molecular  weight marker; C, negative control; F1, nontransgenic kidney  control; S16, internal control (103  nt). (b) Demonstration of linearity of PCR amplification. Total  kidney cDNA (0.1 to 4 μl) aliquots were amplified with primers  specific for c-fos and S16 as internal control. For semiquantitative  evaluation, acrylamide gels (bottom) were scanned by computerized densitometer and graphed  (top). SBF (filled squares); S16  (open squares).
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Figure 3: Expression analysis of the SBF transgenic mice. (a) RT-PCR expression analysis of SBF (234-bp fragment) in 4 different transgenic lines (SBF 38, 19, 29, and 20) shows strong expression of the SBF construct in kidney (K) and spleen (S), but not in liver (Li), heart (H), lung (Lu), or brain (B), shown for SBF 38 only). SBF38 shows high expression of the transgene in kidney and spleen and weak expression in lung and brain. M, molecular weight marker; C, negative control; F1, nontransgenic kidney control; S16, internal control (103 nt). (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1 to 4 μl) aliquots were amplified with primers specific for c-fos and S16 as internal control. For semiquantitative evaluation, acrylamide gels (bottom) were scanned by computerized densitometer and graphed (top). SBF (filled squares); S16 (open squares).

Mentions: Fig. 3 a shows RT-PCR analysis of the SBF transgene in the various organs (including kidney, liver, spleen, heart, lung, and brain). The highest levels of SBF transgene expression were obtained in kidney, where levels were demonstrated to be within the linear range (Fig. 3 b). Some expression was also occasionally detectable in spleen and lung, with little or undetectable expression in other organs (Fig. 3 a). This particular organ distribution of transgene expression closely resembles that described previously for SBM mice (35), and it is likely that the transgene is preferentially targeted to the kidney due to common regulatory elements with SBM. Furthermore, to determine more precisely the localization of the c-fos transgene in SBF kidneys, we have carried out in situ hybridization. Adult transgenic SBF kidneys demonstrated high expression of the transgene in tubular epithelial cells throughout the kidney, with particularly intense signal over the medullary collecting tubules (Fig. 4 a). This cellular localization is very similar to that observed for the c-myc transgene. No expression was detected in renal tissue of non transgenic controls (C57BL/6 × CBA)F1 (Fig. 4 b). Furthermore, c-fos expression was also investigated in the SBM mouse kidneys (Fig. 4 c); the absence of signal indicates that c-fos is not induced in the SBM c-myc–dependent cystogenic pathway.


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)

Expression analysis  of the SBF transgenic mice. (a)  RT-PCR expression analysis of  SBF (234-bp fragment) in 4 different transgenic lines (SBF 38,  19, 29, and 20) shows strong expression of the SBF construct in  kidney (K) and spleen (S), but not  in liver (Li), heart (H), lung (Lu),  or brain (B), shown for SBF 38  only). SBF38 shows high expression of the transgene in kidney  and spleen and weak expression  in lung and brain. M, molecular  weight marker; C, negative control; F1, nontransgenic kidney  control; S16, internal control (103  nt). (b) Demonstration of linearity of PCR amplification. Total  kidney cDNA (0.1 to 4 μl) aliquots were amplified with primers  specific for c-fos and S16 as internal control. For semiquantitative  evaluation, acrylamide gels (bottom) were scanned by computerized densitometer and graphed  (top). SBF (filled squares); S16  (open squares).
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Related In: Results  -  Collection

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Figure 3: Expression analysis of the SBF transgenic mice. (a) RT-PCR expression analysis of SBF (234-bp fragment) in 4 different transgenic lines (SBF 38, 19, 29, and 20) shows strong expression of the SBF construct in kidney (K) and spleen (S), but not in liver (Li), heart (H), lung (Lu), or brain (B), shown for SBF 38 only). SBF38 shows high expression of the transgene in kidney and spleen and weak expression in lung and brain. M, molecular weight marker; C, negative control; F1, nontransgenic kidney control; S16, internal control (103 nt). (b) Demonstration of linearity of PCR amplification. Total kidney cDNA (0.1 to 4 μl) aliquots were amplified with primers specific for c-fos and S16 as internal control. For semiquantitative evaluation, acrylamide gels (bottom) were scanned by computerized densitometer and graphed (top). SBF (filled squares); S16 (open squares).
Mentions: Fig. 3 a shows RT-PCR analysis of the SBF transgene in the various organs (including kidney, liver, spleen, heart, lung, and brain). The highest levels of SBF transgene expression were obtained in kidney, where levels were demonstrated to be within the linear range (Fig. 3 b). Some expression was also occasionally detectable in spleen and lung, with little or undetectable expression in other organs (Fig. 3 a). This particular organ distribution of transgene expression closely resembles that described previously for SBM mice (35), and it is likely that the transgene is preferentially targeted to the kidney due to common regulatory elements with SBM. Furthermore, to determine more precisely the localization of the c-fos transgene in SBF kidneys, we have carried out in situ hybridization. Adult transgenic SBF kidneys demonstrated high expression of the transgene in tubular epithelial cells throughout the kidney, with particularly intense signal over the medullary collecting tubules (Fig. 4 a). This cellular localization is very similar to that observed for the c-myc transgene. No expression was detected in renal tissue of non transgenic controls (C57BL/6 × CBA)F1 (Fig. 4 b). Furthermore, c-fos expression was also investigated in the SBM mouse kidneys (Fig. 4 c); the absence of signal indicates that c-fos is not induced in the SBM c-myc–dependent cystogenic pathway.

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