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, 43 (1), 18-30

Hepatomegaly in Transgenic Mice Expressing the Homeobox Gene Cux-1

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Hepatomegaly in Transgenic Mice Expressing the Homeobox Gene Cux-1

Gregory B Vanden Heuvel et al. Mol Carcinog.

Abstract

Cux-1 is a member of a family of homeobox genes structurally related to Drosophila Cut. Mammalian Cut proteins function as transcriptional repressors of genes specifying terminal differentiation in multiple cell lineages. In addition, mammalian Cut proteins serve as cell-cycle-dependent transcriptional factors in proliferating cells, where they function to repress expression of the cyclin kinase inhibitors p21 and p27. Previously we showed that transgenic mice expressing Cux-1 under control of the CMV immediate early gene promoter develop multiorgan hyperplasia. Here we show that mice constitutively expressing Cux-1 exhibit hepatomegaly correlating with an increase in cell proliferation. In addition, the increase in Cux-1 expression in transgenic livers was associated with a decrease in p21, but not p27, expression. Within transgenic livers, Cux-1 was ectopically expressed in a population of small cells, but not in mature hepatocytes, and many of these small cells expressed markers of proliferation. Transgenic livers showed an increase in alpha-smooth muscle actin, indicating activation of hepatic stellate cells, and an increase in cells expressing chromogranin-A, a marker for hepatocyte precursor cells. Morphological analysis of transgenic livers revealed inflammation, hepatocyte swelling, mixed cell foci, and biliary cell hyperplasia. These results suggest that increased expression of Cux-1 may play a role in the activation of hepatic stem cells, possibly through the repression of the cyclin kinase inhibitor p21.

Figures

Figure 1
Figure 1
Increased liver/body weight ratios in transgenic mice. Three male and three female CMV/Cux-1 mice and wild-type littermates were weighed at 8, 10, and 14 months. The livers from these mice were isolated and weighed. The data are plotted as the average liver/body weight ratio for transgenic (filled diamonds) and wild type (open boxes) mice. Standard deviation is indicated.
Figure 2
Figure 2
Expression of Cux-1, p21, and p27 protein in livers from wild type and transgenic mice. Fifty micrograms of nuclear extract prepared from 8-month-old transgenic and wild type livers was subjected to SDS–PAGE and transferred to nitrocellulose membranes. The presence of Cux-1, p21, p27, and β-actin (as a loading control) was detected by Western blot analysis as described in the ‘‘Materials and Methods.’’ Cux-1 protein was elevated in the transgenic livers compared to wild type. p21 expression was decreased in the transgenic livers, compared to wild type, while p27 expression was unchanged.
Figure 3
Figure 3
Increased proliferation in transgenic livers. The proportion of proliferating cells is increased in livers from CMV/Cux-1 mice (A). The data are plotted as the number of Ki67 positive cells per high power field. Ki67 positive cells were counted in five high power fields from four different animals at each time point. Cell counts in transgenic animals were obtained from extrafocal regions. Sections are shown from 10-month-old wild type liver (B) and extra-focal region of transgenic liver (C). Standard deviation is indicated by error bars. Original magnification: 100× (Bar, 5 µm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 4
Figure 4
Increased proliferation of hepatocyte precursor cells in transgenic livers. Wild type (A, C, E) and transgenic (B, D, F) liver sections from 8-month-old mice were labeled with antibodies directed against Cux-1 (A, B), chromogranin-A (C, D), or PCNA (E, F). Cux-1 was ectopically expressed in small cells (arrows in B) between the hepatocytes in transgenic livers, but was not detected in wild type livers (A). Transgenic livers showed an increase in the number of cells labeled positively for chromogranin-A (arrows in D), a marker for hepatocyte precursor cells. Inset in (D) shows that chromogranin-A was detected in cells morphologically resembling hepatocytes, indicating differentiation to the hepatocyte lineage. No chromogranin-A positive cells were observed in the wild type liver (C). The small, fibroblastic shaped cells, but not the hepatocytes, labeled positively for PCNA (arrows in F), indicating that they are proliferating. No PCNA positive cells were detected in the wild type liver (E). Original magnification: 400× (Bar, 50 µm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 5
Figure 5
Increased expression of α-smooth muscle actin in transgenic livers. Light micrographs of wild type (A) and transgenic livers (B) from 8-month-old mice labeled with antibodies to α-smooth muscle actin. α-smooth muscle actin was found only in the blood vessel wall of liver isolated from an 8-month-old wild type mouse (A). α-smooth muscle actin staining is observed in cells throughout the parenchyma of liver isolated from an 8-month-old transgenic mouse (B). Original magnification: 200×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 6
Figure 6
Gross appearance of liver lesions in CMV/Cux-1 transgenic mice. Beginning at 8 months of age we observed gross alterations in the appearance of transgenic livers, ranging from small discolorations (arrow) to large tumors (arrowhead). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 7
Figure 7
Progression of liver lesions in CMV/Cux-1 transgenic mice. Light micrographs of wild type (A) and transgenic livers (B-H). At 6 months of age, transgenic livers (B) are indistinguishable from wild type livers (A). At 8 months of age, non-suppurative inflammation (C) and fatty cell change (D) are observed in transgenic livers. At 10 months of age, multifocal lesions are evident in transgenic livers (asterisks in E). Higher magnification reveals that these are of the mixed cell type (F). At 14 months of age, hepatocellular carcinoma is present in transgenic livers (G). Biliary hyperplasia is also observed (H). Original magnification: (A–D, F–H) 200× (Bar, 100 µm). (E) 13×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 8
Figure 8
Fatty cell change in transgenic livers. Wild type (A) and transgenic (B, C) livers were stained with Oil-red-O (A, B) or periodic acid schiff (C) stains. Oil-red-O stain reveals fatty change in transgenic livers (arrows in B) compared to wild type, and an increase in PAS positive material in the transgenic livers. Original magnification: 200× (Bar, 100 µm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 9
Figure 9
Ectopic expression of Cux-1 increases in lesion progression. Localization of Cux-1 protein in 8-, 10-, and 14-month-old transgenic livers showing fatty cell change (A, B), mixed cell foci (C, D), and hepatocellular carcinoma (E, F). Small Cux-1 positive cells were observed throughout the 8-month-old transgenic livers in areas of fatty cell change (A, and arrows in B). In some areas of 10-month-old transgenic liver, corresponding to mixed cell foci, high concentrations of Cux-1 positive cells were observed (C). Higher magnification revealed that the Cux-1 positive cells corresponded to only small cells (arrows in D), while hepatocytes were not labeled for Cux-1. In a section of 14-month-old transgenic liver corresponding to hepatocellular carcinoma, Cux-1 positive cells were observed (E), and higher magnification revealed that these cells were small cells (arrows in F), but not hepatocytes. Original magnification: (A, C, E) 100× (Bar, 100 µm). (B, D, F) 400× (Bar, 50 µm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 10
Figure 10
Co-expression of Cux-1 and PCNA in perivascular cells. Adjacent sections of the same liver isolated from CMV/Cux-1 transgenic were stained with PAS (A), labeled with antibodies directed against Cux-1 (B), and labeled with antibodies directed against PCNA (C), showing that cells ectopically expressing Cux-1 were highly proliferative. Original magnification: 200×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 11
Figure 11
Survey of white blood cells in wild type and transgenic liver. Sections of wild type (A) and transgenic (B) livers isolated from 8-month-old mice were labeled with antibodies directed against CD45 (leukocyte common antigen) and CD90.2 to detect all white blood cells. Positive staining cells were detected throughout both livers and cell counting revealed no significant differences in white blood cell number between wild type and transgenic. Labeling with antibodies directed against α-smooth muscle actin (C) showed that the perivascular cells were myofibroblasts. Original magnification: 200×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 12
Figure 12
Expression of Cux-1 in liver tumor of CMV/Cux-1 mouse corresponds to α-smooth muscle actin expression. Adjacent sections from the same tumor were labeled with antibodies directed against Cux-1 (A), α-smooth muscle actin (B), and CD45/CD90.2. Cux-1 positive cells show a similar pattern as α-smooth muscle actin positive cells (Arrows in A and B). In contrast, there are relatively few CD45/CD90.2 positive cells in the tumor. Original magnification: 200×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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