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Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

Robert MC, Furlan G, Rosso N, Gambaro SE, Apitsionak F, Vianello E, Tiribelli C, Gazzin S - PLoS ONE (2013)

Bottom Line: The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001).This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway.These two phenomena might be intimately connected.

View Article: PubMed Central - PubMed

Affiliation: Fondazione Italiana Fegato (Italian Liver Foundation, Centro Studi Fegato), Trieste, Italy.

ABSTRACT
Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

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Representative Western blot and protein relative expression of p18-Cyclin E, Parp-1, cParp-1 and cCasp3.A) Representative Western blot, and B) protein relative quantification. cParp-1: cleaved Parp-1. cCasp3: active caspase3 □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.
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pone-0079073-g004: Representative Western blot and protein relative expression of p18-Cyclin E, Parp-1, cParp-1 and cCasp3.A) Representative Western blot, and B) protein relative quantification. cParp-1: cleaved Parp-1. cCasp3: active caspase3 □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.

Mentions: Because the increased expression of Cyclin E was unexpected and, apparently, opposite to the concept of cell cycle arrest, we decided to better analyse its up-regulation. For this reason, we used the M-20 antibody (C-terminal epitope) which recognize the p18-Cyclin E proteolytic fragment (Figure 4A), with known pro-apoptotic properties [63–65]. As shown in Figure 4B, the p18-Cyclin E level was increased by about 67% (p < 0.05). In agreement, Parp and cleaved Parp-1 (cParp-1, Figure 4A), a marker of early apoptosis [66,67], were significantly increased in jj animals by 38% and 54%, respectively (both p < 0.01) as compared to control (JJ) animals (Figure 4B). Under standard Western blot conditions, the protein expression of cCasp3 was only detectable in jj animals (data not shown). In order to perform the quantification, we obtained the signal by exposing the film longer. Under these conditions, we observed a cCasp3 amount double in hyperbilirubinemic pups vs. the JJ control (p < 0.01) (Figure 4B).


Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

Robert MC, Furlan G, Rosso N, Gambaro SE, Apitsionak F, Vianello E, Tiribelli C, Gazzin S - PLoS ONE (2013)

Representative Western blot and protein relative expression of p18-Cyclin E, Parp-1, cParp-1 and cCasp3.A) Representative Western blot, and B) protein relative quantification. cParp-1: cleaved Parp-1. cCasp3: active caspase3 □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815147&req=5

pone-0079073-g004: Representative Western blot and protein relative expression of p18-Cyclin E, Parp-1, cParp-1 and cCasp3.A) Representative Western blot, and B) protein relative quantification. cParp-1: cleaved Parp-1. cCasp3: active caspase3 □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.
Mentions: Because the increased expression of Cyclin E was unexpected and, apparently, opposite to the concept of cell cycle arrest, we decided to better analyse its up-regulation. For this reason, we used the M-20 antibody (C-terminal epitope) which recognize the p18-Cyclin E proteolytic fragment (Figure 4A), with known pro-apoptotic properties [63–65]. As shown in Figure 4B, the p18-Cyclin E level was increased by about 67% (p < 0.05). In agreement, Parp and cleaved Parp-1 (cParp-1, Figure 4A), a marker of early apoptosis [66,67], were significantly increased in jj animals by 38% and 54%, respectively (both p < 0.01) as compared to control (JJ) animals (Figure 4B). Under standard Western blot conditions, the protein expression of cCasp3 was only detectable in jj animals (data not shown). In order to perform the quantification, we obtained the signal by exposing the film longer. Under these conditions, we observed a cCasp3 amount double in hyperbilirubinemic pups vs. the JJ control (p < 0.01) (Figure 4B).

Bottom Line: The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001).This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway.These two phenomena might be intimately connected.

View Article: PubMed Central - PubMed

Affiliation: Fondazione Italiana Fegato (Italian Liver Foundation, Centro Studi Fegato), Trieste, Italy.

ABSTRACT
Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

Show MeSH
Related in: MedlinePlus