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H C Bisgaard

Publications and source records attributed to H C Bisgaard.

At least 19 recordsLinked to original sources
PubMed2001

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system. In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis.

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.

14-3-3 Proteins↗
PubMed2000

Studies on hepatic gene expression in different liver regenerative models.

We have investigated the expression of several growth-related genes in the liver after partial hepatectomy in three experimental models: normal, Dexamethasone-pretreated, and hypophysectomized rats. Dexamethasone and hypophysectomy resulted in a delay in the peak of cell replication in 6 and 18 h, respectively, when compared to the normal animals. TGFalpha mRNA expression was shifted together with the DNA synthesis, but the expression of c-myc, c-fos, c-jun, HGF, TGFbeta1, IL1beta did not delay. This result suggests that liver-derived TGFalpha but not the other factors are important in the timing of the proliferative response after partial hepatectomy.

Animals↗
PubMed2000

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

Alkylating Agents↗
PubMed1999

Interaction between ellagic acid and calf thymus DNA studied with flow linear dichroism UV-VIS spectroscopy.

The interaction between ellagic acid and DNA has been characterized with respect to the geometry of the ellagic acid-DNA complex, and the active form of ellagic acid has been identified. Optical spectroscopic methods have been employed to examine the interaction between double-stranded calf thymus DNA and ellagic acid in low-ionic-strength aqueous solutions at pH values of 5.5, 7.0, and 8. 8. Based on normal absorption titration and flow linear dichroism experiments, it is confirmed that the neutral form of ellagic acid present at pH 5.5 binds to double-stranded DNA. It is found that the plane of the ellagic acid chromophore is positioned at an angle relative to the DNA helix axis, which is in accordance with intercalation of ellagic acid in DNA. It is concluded that at higher values of pH no or a very limited amount of ellagic acid binds to DNA. These results prove that the direct interaction between ellagic acid and DNA must be taken into account when evaluating the mechanism underlying the observed biological effects of this plant phenol.

Animals↗
PubMed1999

Modulation of the gene network connected to interferon-gamma in liver regeneration from oval cells.

Suppression subtractive hybridization was used to clone genes associated with proliferation of oval cells in rat liver regenerating after a 70% partial hepatectomy combined with the feeding of 2-acetylaminofluorene. A subset of the identified genes comprised interferon-gamma receptor alpha subunit (IFN-gammaRalpha), gp91phox, interleukin-1beta (IL-1beta), lymphocyte function-associated molecule-1alpha (LFA-1), eukaryotic initiation factor-2-associated 67-kd protein (eIF-2-associated 67-kd protein), and alpha-fetoprotein, which constitute part of the cellular program modulated by IFN-gamma. Therefore, expression analysis performed by Northern blotting and immunohistochemistry were extended to include IFN-gamma, the IFN-gamma receptor beta subunit (IFN-gammaRbeta), three secondary response genes induced by interaction of IFN-gamma with IFN-gamma receptor complexes, ie, IL-1beta-converting enzyme (ICE), intercellular adhesion molecule-1 (ICAM-1), and urokinase-type plasminogen activator receptor (uPAR), and a cytokine inducing IFN-gamma expression, ie, interleukin-18 (IL-18). The Northern blot analysis showed that all examined genes were modulated when progenitor-like oval cells were activated and recruited for liver regeneration. Immunohistochemistry localized the subunits of the IFN-gamma receptor complex, IFN-gammaRalpha and IFN-gammaRbeta, the secondary response genes uPAR and ICAM-1, the IFN-gamma-inducing factor IL-18, and ICE to the ductular structures of oval cells. In contrast, during liver regeneration after a 70% partial hepatectomy, only modulation of IL-1beta and ICE was observed. Our results, therefore, indicate that IFN-gamma-mediated events may be particularly important when cells in the bile ductules must respond to liver damage by production of ductular oval cells.

2-Acetylaminofluorene↗
PubMed1998

Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells.

The proteolytic cascade involving plasminogen activators and plasmin appears to have an important function in tissue regeneration. We have investigated the expression and cellular localization of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) as well as plasminogen activation in rat liver regeneration by recruitment of progenitor (oval) cells. Using a model in which surgical partial hepatectomy is combined with feeding of 2-acetylaminofluorene (2-AAF) to induce liver regeneration by proliferation and differentiation of oval cells, expression of uPA, uPAR, and PAI-1 was detected by immunohistochemistry mainly in the duct-like formations of expanding oval cells. Plasminogen activation, as assessed by direct zymography on frozen liver sections, was located over the expanding oval cell populations but not over mature hepatocytes. Plasminogen activation was not detected in control liver. Expression of uPA, uPAR, and PAI-1, as assessed by immunohistochemical and Northern blot analyses, was also observed, when cells located in and in close proximity to the bile epithelial structures were activated to enter DNA-synthesis in response to 2-AAF, and after in vivo infusion of various growth factors. Given the physiologic function of plasminogen activation in fibrinolysis, and plasminogen activators in activation of latent growth factors, the selective expression of the plasminogen activator/plasmin proteolytic cascade in oval cells expanding during liver regeneration in response to the combination of 2-AAF and partial hepatectomy, may confer a proliferative advantage to these cell populations in an extracellular matrix containing both fibrin and latent growth factors.

2-Acetylaminofluorene↗
PubMed1997

Spontaneous metastasis of rat liver epithelial cells transformed with v-raf and v-raf/v-myc: association with different phenotypic properties.

Cloned v-raf, v-raf/v-myc, and spontaneously transformed rat liver epithelial (RLE) cell lines were examined for meastatic capability in nude mice, using the LacZ gene as a marker for quantitation of micrometastases. Six cloned lines (R3611-T lines) derived from nude mouse xenografts of the v-raf transformed R3611-3 cells displayed variable metastatic capabilities. Three of six subcutaneously inoculated R3611-TlacZ lines produced spontaneous lung metastasis in nude mice. One of the lines, R3611-T2lacZ was highly efficient at metastatic conversion and produced more lung colonies than a faster growing v-raf/v-myc-transformed RJ2-14lacZ line. The spontaneously transformed RLElacZ line (C4T) was nonmetastatic, although it produced larger subcutaneous tumors than the metastatic R3611-T2lacZ line. Metastatic conversion correlated with upregulation of urokinase-type plasminogen activator receptor RNA expression and downregulation of plasminogen activator inhibitor-1, collagen alpha1 (I), and cytokeratin 14 (K14) RNA expression. These findings indicate that proteolytic activities associated with plasminogen activation play a role in the metastatic development in this model. Decreased production of extracellular proteins and cytoskeletal changes associated with lack of K14 expression are also likely to have contributed to the metastatic conversion of the RLE transformants.

Animals↗
PubMed1996

Proliferation, apoptosis, and induction of hepatic transcription factors are characteristics of the early response of biliary epithelial (oval) cells to chemical carcinogens.

In this study, we used [3H]thymidine labeling of newly synthesized DNA to examine the earliest effects of 2-acetylaminofluorene (2-AAF) on the mitotic activation of cells in the adult rat liver, and in situ hybridization analysis to study the expression of three transcription factors (HNF1 beta, HNF3 gamma, and HNF4), and two of the genes (alpha-fetoprotein [AFP] and albumin) regulated by these factors. A low dose of 2-AAF (and its analogs, 2-AF [2-aminofluorene] and N-OH-2-AAF) elicited a mitogenic response in ductal cells and nondescript periductular cells within 24 hours after administration. The compounds also induced the expression of HNF1 beta, HNF3 gamma, AFP, and albumin in ductal structures but had no detectable effect of HNF4 expression. In contrast, initiation of bile duct proliferation by ligation of the common bile duct had no effect on the expression of these genes in ductal cells. In addition to inducing a mitogenic response, 2-AAF resulted in increased numbers of apoptotic cells in the portal areas, a process that contributed to overall retention of liver morphology. Our results demonstrate that 2-AAF and some of its analogs can elicit a specific mitogenic response and induce expression of the "establishment" transcription factors, HNF1 beta and HNF3 gamma, in ductal cells. Our data provide further support of a precursor-product relationship between "stem-like" cells located in ductal structures, oval cells, and hepatocytes.

2-Acetylaminofluorene↗
PubMed1996

In vivo infusion of growth factors enhances the mitogenic response of rat hepatic ductal (oval) cells after administration of 2-acetylaminofluorene.

Expression of several growth factors is elevated in rat liver, after induction of oval cell proliferation by chemical carcinogens. However, the exact roles played by individual factors are not defined. We infused and examined the effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on the proliferation of ductal and periductal cells after their activation with 2-acetylaminofluorene (2-AAF). Furthermore, we included studies on urokinase-type plasminogen activator (uPA), because Northern blot analysis showed a strong coincidence of uPA expression with oval cell proliferation. Low doses of 2-AAF were used to activate ductal and periductal cells, whereafter growth factors were infused. Infusion of EGF, HGF, uPA, or any combination thereof for up to 7 days resulted in increased numbers of [3H]thymidine-labeled ductal and periductal cells expanding into the liver acinus. Although the growth factors all increased the number of labeled cells, they preferentially acted on different cell populations. Although exposure to 2-AAF alone or combined with infusion of HGF resulted in proliferation of almost equal numbers of ductal and Ito cells, infusion of EGF and any combination hereof resulted in 75% to 80% of labeled cells having a ductal phenotype. Also, infusion of EGF and HGF resulted in decreased numbers of cells undergoing apoptosis in response to 2-AAF. Our results demonstrate that, although 2-AAF acts as a mitogenic stimulus for ductal and periductal cells, growth factors are necessary for survival, motility, and expansion of these cells into the liver acini.

2-Acetylaminofluorene↗
PubMed1996

Hepatic regeneration. The role of regeneration in pathogenesis of chronic liver diseases.

Hepatic necrosis is a common reaction to liver injury of various etiologies. The response is regeneration. As reviewed earlier, reconstitution of liver mass may proceed via two mechanisms: (1) re-entry of surviving, functionally intact differentiated liver cells into the cell cycle, where they may remain for several rounds of replication, and (2) recruitment of hepatic progenitor cells, whereby the liver mass is replaced by extensive proliferation and differentiation of more primitive cell types. Although both mechanisms appear to share a number of regulatory factors, distinct differences exist that are reflected in the complex and intricate networks of interacting cells, cytokines, and ECM molecules constituting the regenerative process. The development of liver fibrosis or cirrhosis is probably an unwanted but frequent byproduct of the regenerative process, similar to scar formation in any other tissue following extensive damage. Although intensive research in recent years has yielded a wealth of information about regenerative processes, a better understanding of the elements regulating the regenerative process is crucial for effective intervention to prevent or minimize fibrosis while providing optimal conditions for regeneration. Because our only experimental tool is observation in human studies, we must continue the use of experimental animal models including those of transgenic mice to further elucidate the complex interactions of cytokines, ECM, and target cells in the development of liver fibrogenesis, cirrhosis, and cancer.

Animals↗
PubMed1994

Modulation of keratin 14 and alpha-fetoprotein expression during hepatic oval cell proliferation and liver regeneration.

Keratin 14 (K14) expression has recently been demonstrated in cell lines of non-parenchymal hepatic origin (Bisgaard et al., 1993, Mol. Carcinog., 7:60-66; Bisgaard et al., 1991, J. Cell. Physiol., 147:333-343). These cell lines are thought to represent a progeny of a dormant stem cell compartment present in the adult rat liver, which may participate in the restoration of the liver mass after experimental liver injury. Utilizing a combination of 2-acetylaminofluorene (2-AAF) administration and partial hepatectomy to activate liver regeneration by proliferation of oval cells, we examined the modulation of K14 as well as alpha-fetoprotein (AFP) expression in proliferating oval cells and lineages hypothesized to be derived herefrom. We showed by Northern blot and in situ hybridization analyses that K14 and AFP transcripts were initially accumulating in epithelial cells located in subsets of ductal structures in the portal areas. As oval cells infiltrated the liver parenchyma, K14 transcripts were detected in oval cells, in foci of small basophilic hepatocytes, and in structures resembling glandular intestinal-type epithelium. AFP was expressed in oval cells, and at low but detectable levels in foci of basophilic hepatocytes, but not in glandular intestinal-type epithelium. Neither K14 nor AFP transcripts were detected in bile ducts or mature hepatocytes at any time during oval cell proliferation and reconstitution of the liver mass. To further study the modulation of K14 and AFP expression we utilized an in vitro model in which spontaneous transformation of rat liver epithelial (RLE) cells appeared to mimic the process of early differentiation along the hepatic lineage in vivo. We demonstrated that undifferentiated RLE cells at a late passage expressed K14 and vimentin, whereas transformation and differentiation to hepatoblast-like progeny resulted in an abrogation of K14 and vimentin expression and an induction of K18 and AFP. We propose that K14 and AFP are sequentially modulated in subpopulations of oval cells involved in the ongoing reconstitution of the liver mass.

2-Acetylaminofluorene↗
PubMed1994

Phenotypic modulation of keratins, vimentin, and alpha-fetoprotein in cultured rat liver epithelial cells after chemical, oncogene, and spontaneous transformation.

Several lines of evidence have indicated that rat liver epithelial (RLE) cell lines may be related to a dormant stem cell compartment in the liver in vivo. We have demonstrated that keratin 14 (K14) is expressed together with vimentin in undifferentiated RLE cells. However, upon spontaneous transformation and differentiation to hepatoblast-like progeny the expression of these intermediate filaments (IF) is abrogated, while expression of another set of genes, among others keratin 18 (K18) and alpha-fetoprotein (AFP), is induced (Bisgaard et al., 1994, J. Cell. Physiol., in press). To better understand the mechanisms underlying IF expression during transformation and differentiation of RLE cells we examined the expression and regulation of IFs in clonal cell lines of chemically, oncogene, and spontaneously transformed RLE cells and their resulting tumors. These clonal lines provided a wide variety of tumor phenotypes including trabecular, solid and tubular adenocarcinomas, undifferentiated carcinomas, and spindle cell carcinomas. Northern blot analysis of the cell lines confirmed the differential expression of IF mRNAs. While keratin 8 (K8) was expressed at similar steady-state levels in all cell lines, K14 and vimentin but not K18 were expressed in the majority of cell lines chemically transformed with aflatoxin B1 or by transduction of oncogenes. In contrast, cell lines transformed spontaneously by prolonged passage in vitro expressed K18, while K14 and vimentin were absent. The keratin expression pattern in vitro was retained in the majority of the resulting tumors. However, the keratins expressed in vitro did not accurately predict the tumor phenotype in vivo. In particular, in tumors typed morphologically as adenocarcinomas, the keratin pair typically expressed in chemically transformed tumor cells was K8/K14, whereas K8/K18 was expressed in the tumors derived from spontaneously transformed cell lines. Finally we showed by nuclear run-on and in vitro translation analyses that the expression of K14, K18, and vimentin in transformed RLE cell lines was regulated at the transcriptional level, whereas that of K8 appeared to be posttranslational. These findings suggest that events controlling the differential expression of IF genes are involved in the processes leading to transformation and differentiation of the RLE cell lines. We conclude that the transformed RLE cell lines provide a valuable model to further examine the regulatory mechanisms involved in hepatic differentiation of undifferentiated "progenitor-like" RLE cells.

Animals↗
PubMed1994

Expression of hepatic transcription factors during liver development and oval cell differentiation.

The oval cells are thought to be the progeny of a liver stem cell compartment and strong evidence now exists indicating that these cells can participate in liver regeneration by differentiating into different hepatic lineages. To better understand the regulation of this process we have studied the expression of liver-enriched transcriptional factors (HNF1 alpha and HNF1 beta, HNF3 alpha, HNF3 beta, and HNF3 gamma, HNF4, C/EBP, C/EBP beta, and DBP) in an experimental model of oval cell proliferation and differentiation and compared the expression of these factors to that observed during late stages of hepatic ontogenesis. The steady-state mRNA levels of four (HNF1 alpha, HNF3 alpha, HNF4, and C/EBP beta) "liver-enriched" transcriptional factors gradually decrease during the late period of embryonic liver development while three factors (HNF1 beta, HNF3 beta, and DBP) increase. In the normal adult rat liver the expression of all the transcription factors are restricted to the hepatocytes. However, during early stages of oval cell proliferation both small and large bile ducts start to express HNF1 alpha and HNF1 beta, HNF3 gamma, C/EBP, and DBP but not HNF4. At the later stages all of these factors are also highly expressed in the proliferating oval cells. Expression of HNF4 is first observed when the oval cells differentiate morphologically and functionally into hepatocytes and form basophilic foci. At that time the expression of some of the other factors is also further increased. Based on these data we suggest that the upregulation of the "establishment" factors (HNF1 and -3) may be an important step in oval cell activation. The high levels of these factors in the oval cells and embryonic hepatoblasts further substantiates the similarity between the two cell compartments. Furthermore, the data suggest that HNF4 may be responsible for the final commitment of a small portion of the oval cells to differentiate into hepatocytes which form the basophilic foci and eventually regenerate the liver parenchyma.

Animals↗
PubMed1993

Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV-6.

We have recently reported that cell lines of nonparenchymal origin isolated from rat liver and pancreas, which have been suggested to be the progeny of a facultative stem cell compartment in vivo, express an unusual combination of keratins (K). These cell lines express K8 and K14 but not K18 and K5, their normal partners in filament formation (Bisgaard HC, Thorgeirsson SS, J Cell Physiol 147:333-343, 1991). However, upon spontaneous transformation and differentiation toward a hepatoblastlike progeny, K14 expression is abrogated and replaced by expression of K18 (Wirth et al., Electrophoresis 13:305-332, 1992). In the study presented here, we confirmed by protein sequence analysis that K14 was a major component of the intermediate filaments in a nonparenchymal cell line of hepatic origin. Immunocytochemical analysis of the cells in monolayer demonstrated that K8 as well as K14 were incorporated in the cellular cytoskeleton. Further analysis by immunoprecipitation showed that filament complexes were formed between K8 and K14 as atypical partners. Thus, we concluded that in some nonparenchymal cell lines isolated from rat liver, K8 and K14 form a major intermediate filament network. Finally, we showed that an antibody widely used in studies of the cell lineages of hepatic and pancreatic tissues and their neoplasms, the mouse monoclonal antibody OV-6, recognizes a common epitope in K14 and K19.

Amino Acid Sequence↗
PubMed1992

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.

Actins↗