In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it pos... more In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it possible to distinguish a sinusoidal, a lateral and a canalicular domain. The conditions that determine hepatocyte plasma membrane polarity have been investigated in vitro, using three monoclonal antibodies directed against integral membrane proteins, which were characteristic of each domain. The localization of the three antigens was studied by immunolabelling of hepatocytes isolated from adult rat liver, primary monolayer cultures and rat hepatoma cell lines. When hepatocytes were isolated, the three antigens spread over the entire cell surface. The lateral antigen redistributed at lateral sites as soon as cell-cell contacts were established, 4 h after the beginning of primary culture. The sinusoidal and canalicular antigens became asymmetrically distributed after 48 h of primary culture, after the formation of bile canaliculus-like structures. In most of the hepatoma lines studied, the t...
In order to analyze the mechanisms implicated in the expression of differentiated functions durin... more In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functi...
PFIC2 is due to mutations in ABCB11 encoding BSEP, the canalicular bile salt export pump of hepat... more PFIC2 is due to mutations in ABCB11 encoding BSEP, the canalicular bile salt export pump of hepatocyte. In some PFIC2 patients with missense mutations, BSEP is not detected at the canaliculus due to mistrafficking of BSEP mutants. In vitro, chaperone drugs such as 4-phenylbutyrate (4-PB) have been shown to partially correct mistrafficking. Four PFIC2 patients harboring at least one missense mutation (p.G982R, p.R1128C, p.T1210P) were treated orally with 4-PB and followed prospectively. Patient mutations were reproduced in a Bsep-GFP plasmid. Cellular localization of the resulting Bsep mutants was studied in a hepatocellular line (Can 10) and effects of treatment with 4-PB and/or ursodeoxycholic acid (UDCA) were assessed. In Can 10 cells, Bsep mutants were detected in the endoplasmic reticulum instead of at the canalicular membrane. Treatment with 4-PB and with UDCA partially corrected Bsep mutants targeting. With 4-PB, we observed in all patients a decrease of pruritus and of serum bile acid concentration and an improvement of serum liver tests. Pathological liver injuries improved and BSEP, which was not detected at the canalicular membrane before treatment, appeared at the canalicular membrane. Bile analyses showed an increase in bile acid concentration with 4-PB. Patient conditions remained stable with a median follow-up of 40 months (range 3-53) and treatment tolerance was good. Conclusion: 4-PB therapy may be efficient in selected patients with PFIC2 due to ABCB11 missense mutations affecting BSEP canalicular targeting. Bile secretion improvement may be due to the ability of 4-PB to retarget mutated BSEP. This article is protected by copyright. All rights reserved.
Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chro... more Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chromosomes show pleiotropic extinction of thirteen out of fifteen hepatic functions examined. Reexpression of the entire group of functions most often occurs in a block, and except for one discordant subclone, correlates with loss of human chromosome 2. The extinguished cells and their reexpressing derivatives have been examined for the expression of seven liver-enriched transcription factors. C/EBP, LAP, DBP, HNF3, and vHNF1 expression are not systematically extinguished in parallel with the hepatic functions. However, HNF1 and HNF4 show a perfect correlation with phenotype: these factors are expressed only in the cells showing pleiotropic reexpression. Since recent evidence indicates that HNF4 controls HNF1 expression, it can be proposed that the HNF4 gene is the primary target of the pleiotropic extinguisher.
Received 7 May 2010; received in revised form 9 July 2010; accepted 13 July 2010. published onlin... more Received 7 May 2010; received in revised form 9 July 2010; accepted 13 July 2010. published online 20 August 2010. ... No abstract is available. To read the body of this article, please view the Full Text online. ... Corresponding Author Information Corresponding author at: ...
Darier&am... more Darier's disease (DD) is an autosomal dominant skin disorder characterized by acantholysis and abnormal keratinization. The gene responsible for DD, ATP2A2 encodes for the sarco/endoplasmic reticulum (ER) Ca2+-ATPase isoform 2 protein. Involucrin, considered as a marker of terminal epidermal differentiation, could be altered in some keratinization disorders including DD. An immunohistochemical staining using anti-involucrin antibody was carried out on 16 DD patients epidermis. Involucrin staining was compared with biopsies from cutaneous lesions of three healthy individuals and of patients with Hailey-Hailey disease (five cases) and Mal de Meleda (four cases). A semi-quantitative analysis was performed in order to evaluate involucrin immunostaining on the basis of intensity, extension and epidermal distribution. The involucrin expression was examined afterward with confocal laser scanning microscopy. In contrast to normal skin, all DD cases showed premature expression of involucrin in the lower epidermal layers in four cases with a strong labeling in both keratinocytes cell membrane and cytoplasm. Other keratinization disorders share premature expression of involucrin but displayed differences in cytoplasm/cell membrane labeling. DD skin displayed a constant immunohistochemical involucrin pattern characterized by both premature expression and a particular cytoplasmic/cell membrane localization distribution.
The ratio of mouse to rat albumin secreted by mouse hepatoma x rat hepatoma hybrid cells is const... more The ratio of mouse to rat albumin secreted by mouse hepatoma x rat hepatoma hybrid cells is constant (of the order of 5.0) irrespective of the total amounts produced. The present results establish for seven independent hybrid clones that the coordination in the ratio of mouse to rat product applies also at the level of accumulation of albumin mRNAs of the two species. The interpretation that coordinate synthesis reflects coordinate transcription of the relevant genes is thus reinforced.
Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been c... more Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been crossed with rat hepatoma cells that secret only albumin, and in relatively small amounts, to investigate the influence of each parental genome upon the expression of serum proteins. All of the ten independent hybrid clones examined produce mouse AFP and both mouse and rat albumin; none produces rat AFP. The absence of production of rat AFP by the hybrids suggests that different mechanisms are involved in the initiation and in the maintenance of expression of this function. The secretion of the three proteins by the hybrid cells is coordinate: Whatever the growth phase (exponential or stationary) and irrespective of the amounts produced over a wide range, the ratio secreted of mouse AFP to mouse albumin is near to one, and that of mouse albumin to rat albumin is near to five. In addition, even though the pattern of protein secretion during the growth cycle of hybrid cells is different from those of both parents, the products of both parental genomes conform to the new hybrid pattern. Finally, some hybrids secrete less of the proteins with increasing numbers of cell generations, yet all three continue to be secreted in coordinate fashion. Since the rates of secretion of serum proteins probably reflect their rates of synthesis, we conclude that coordinate secretion indicates coordinate synthesis, and may reflect coordinate transcription of the relevant genes.
In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it pos... more In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it possible to distinguish a sinusoidal, a lateral and a canalicular domain. The conditions that determine hepatocyte plasma membrane polarity have been investigated in vitro, using three monoclonal antibodies directed against integral membrane proteins, which were characteristic of each domain. The localization of the three antigens was studied by immunolabelling of hepatocytes isolated from adult rat liver, primary monolayer cultures and rat hepatoma cell lines. When hepatocytes were isolated, the three antigens spread over the entire cell surface. The lateral antigen redistributed at lateral sites as soon as cell-cell contacts were established, 4 h after the beginning of primary culture. The sinusoidal and canalicular antigens became asymmetrically distributed after 48 h of primary culture, after the formation of bile canaliculus-like structures. In most of the hepatoma lines studied, the t...
In order to analyze the mechanisms implicated in the expression of differentiated functions durin... more In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functi...
PFIC2 is due to mutations in ABCB11 encoding BSEP, the canalicular bile salt export pump of hepat... more PFIC2 is due to mutations in ABCB11 encoding BSEP, the canalicular bile salt export pump of hepatocyte. In some PFIC2 patients with missense mutations, BSEP is not detected at the canaliculus due to mistrafficking of BSEP mutants. In vitro, chaperone drugs such as 4-phenylbutyrate (4-PB) have been shown to partially correct mistrafficking. Four PFIC2 patients harboring at least one missense mutation (p.G982R, p.R1128C, p.T1210P) were treated orally with 4-PB and followed prospectively. Patient mutations were reproduced in a Bsep-GFP plasmid. Cellular localization of the resulting Bsep mutants was studied in a hepatocellular line (Can 10) and effects of treatment with 4-PB and/or ursodeoxycholic acid (UDCA) were assessed. In Can 10 cells, Bsep mutants were detected in the endoplasmic reticulum instead of at the canalicular membrane. Treatment with 4-PB and with UDCA partially corrected Bsep mutants targeting. With 4-PB, we observed in all patients a decrease of pruritus and of serum bile acid concentration and an improvement of serum liver tests. Pathological liver injuries improved and BSEP, which was not detected at the canalicular membrane before treatment, appeared at the canalicular membrane. Bile analyses showed an increase in bile acid concentration with 4-PB. Patient conditions remained stable with a median follow-up of 40 months (range 3-53) and treatment tolerance was good. Conclusion: 4-PB therapy may be efficient in selected patients with PFIC2 due to ABCB11 missense mutations affecting BSEP canalicular targeting. Bile secretion improvement may be due to the ability of 4-PB to retarget mutated BSEP. This article is protected by copyright. All rights reserved.
Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chro... more Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chromosomes show pleiotropic extinction of thirteen out of fifteen hepatic functions examined. Reexpression of the entire group of functions most often occurs in a block, and except for one discordant subclone, correlates with loss of human chromosome 2. The extinguished cells and their reexpressing derivatives have been examined for the expression of seven liver-enriched transcription factors. C/EBP, LAP, DBP, HNF3, and vHNF1 expression are not systematically extinguished in parallel with the hepatic functions. However, HNF1 and HNF4 show a perfect correlation with phenotype: these factors are expressed only in the cells showing pleiotropic reexpression. Since recent evidence indicates that HNF4 controls HNF1 expression, it can be proposed that the HNF4 gene is the primary target of the pleiotropic extinguisher.
Received 7 May 2010; received in revised form 9 July 2010; accepted 13 July 2010. published onlin... more Received 7 May 2010; received in revised form 9 July 2010; accepted 13 July 2010. published online 20 August 2010. ... No abstract is available. To read the body of this article, please view the Full Text online. ... Corresponding Author Information Corresponding author at: ...
Darier&am... more Darier's disease (DD) is an autosomal dominant skin disorder characterized by acantholysis and abnormal keratinization. The gene responsible for DD, ATP2A2 encodes for the sarco/endoplasmic reticulum (ER) Ca2+-ATPase isoform 2 protein. Involucrin, considered as a marker of terminal epidermal differentiation, could be altered in some keratinization disorders including DD. An immunohistochemical staining using anti-involucrin antibody was carried out on 16 DD patients epidermis. Involucrin staining was compared with biopsies from cutaneous lesions of three healthy individuals and of patients with Hailey-Hailey disease (five cases) and Mal de Meleda (four cases). A semi-quantitative analysis was performed in order to evaluate involucrin immunostaining on the basis of intensity, extension and epidermal distribution. The involucrin expression was examined afterward with confocal laser scanning microscopy. In contrast to normal skin, all DD cases showed premature expression of involucrin in the lower epidermal layers in four cases with a strong labeling in both keratinocytes cell membrane and cytoplasm. Other keratinization disorders share premature expression of involucrin but displayed differences in cytoplasm/cell membrane labeling. DD skin displayed a constant immunohistochemical involucrin pattern characterized by both premature expression and a particular cytoplasmic/cell membrane localization distribution.
The ratio of mouse to rat albumin secreted by mouse hepatoma x rat hepatoma hybrid cells is const... more The ratio of mouse to rat albumin secreted by mouse hepatoma x rat hepatoma hybrid cells is constant (of the order of 5.0) irrespective of the total amounts produced. The present results establish for seven independent hybrid clones that the coordination in the ratio of mouse to rat product applies also at the level of accumulation of albumin mRNAs of the two species. The interpretation that coordinate synthesis reflects coordinate transcription of the relevant genes is thus reinforced.
Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been c... more Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been crossed with rat hepatoma cells that secret only albumin, and in relatively small amounts, to investigate the influence of each parental genome upon the expression of serum proteins. All of the ten independent hybrid clones examined produce mouse AFP and both mouse and rat albumin; none produces rat AFP. The absence of production of rat AFP by the hybrids suggests that different mechanisms are involved in the initiation and in the maintenance of expression of this function. The secretion of the three proteins by the hybrid cells is coordinate: Whatever the growth phase (exponential or stationary) and irrespective of the amounts produced over a wide range, the ratio secreted of mouse AFP to mouse albumin is near to one, and that of mouse albumin to rat albumin is near to five. In addition, even though the pattern of protein secretion during the growth cycle of hybrid cells is different from those of both parents, the products of both parental genomes conform to the new hybrid pattern. Finally, some hybrids secrete less of the proteins with increasing numbers of cell generations, yet all three continue to be secreted in coordinate fashion. Since the rates of secretion of serum proteins probably reflect their rates of synthesis, we conclude that coordinate secretion indicates coordinate synthesis, and may reflect coordinate transcription of the relevant genes.
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