No abstract

Hepatocytes and other cellular elements isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. Analysis of the structures formed demonstrates that the receptor tyrosine kinase ligands HGF and EGF are required for the presence, growth, and phenotypic maturation of the biliary epithelium on the surface of the cultures and for the formation of connective tissue in the cultures. Dexamethasone, in the presence of HGF and EGF, was required for the phenotypic maturation of hepatocytes. The results demonstrate the role of these molecules for the formation and phenotypic maturation of specific histological elements of the liver and suggest roles for these signaling molecules in the formation and structure of the in vivo hepatic architecture.

Corticosteroids, hepatocyte growth factor (HGF), and epidermal growth factor (EGF) play important roles in hepatic biology. We have previously shown that these molecules are required for formation of tissue with specific histology in complex organoid cultures. Dexamethasone suppresses growth and induces hepatocyte maturation; HGF and EGF are needed for formation of the nonepithelial elements. All three are needed for formation of the biliary epithelium. The gene expression patterns by which corticosteroids, HGF, and EGF mediate their effects in hepatic tissue formation are distinct. These patterns affect many gene families and are described in detail. In terms of main findings, dexamethasone induces expression of both HNF4 and C/EBPα, essential transcription factors for hepatocyte differentiation. It suppresses hepatocyte growth by suppressing many molecules associated with growth in liver and other tissues, including IL-6, CXC-chemokine receptor, amphiregulin, COX-2, HIF, etc. HGF and EGF induce all members of the TGF-β family. They also induced multiple CNS-related genes, probably associated with stellate cells. Dexamethasone, as well as HGF and EGF, induces expression of HNF6-β, associated with biliary epithelium formation. Combined addition of all three molecules is associated with mature histology in which hepatocyte and biliary lineages are separate and HNF4 is expressed only in hepatocyte nuclei. In conclusion, the results provide new and surprising information on the gene expression alterations by which corticosteroids, HGF, and EGF exert their effects on formation of hepatic tissue. The results underscore the usefulness of the organoid cultures for generating information on histogenesis, which cannot be obtained by other culture or whole animal models. Gene arraysThree-dimensional cultures Hepatocytes Biliary cells Growth regulation HEPATOCYTES isolated by perfusion of rat liver thelium in the surface of these cultures is in large part derived from hepatocytes undergoing trans-differenwith collagenase contain a small contaminant population of other hepatic cells, such as stellate cells, bilitiation (45). HGF and EGF are required for the formation of the biliary epithelium and the connective ary epithelium, endothelial cells, etc. The primary isolates reorganize to form tissue with recognizable, albeit tissue. The effects on connective tissue are probably mediated by regulation of expression of TGF-β famaltered, hepatic microarchitecture in roller bottle cultures under the influence of insulin, nicotinamide, corily members (46). Dexamethasone, in the absence of HGF and EGF, induces maturation of cells excluticosteroids (dexamethasone), hepatocyte growth factor (HGF), and epidermal growth factor (EGF). Histology sively towards the hepatocytic lineage. Biliary epithelium is absent without addition of HGF or EGF. in these organoid cultures consists of a surface layer of mature biliary epithelium, an intermediate layer ofWhen dexamethasone, HGF, and EGF are all removed, immature hepatocytes present in the cu...

No abstract

No abstract

No abstract

No abstract

8015 Background: The non-classical alkylators DTIC and TMZ exert their cytotoxic effect through methylation of DNA bases. DNA damage is repaired by direct reversal of the O6-MeG lesion by the protein O6-methylguanine-DNA methyltransferase (MGMT/AGT); recognition of alkylation-damage-induced replication errors by the MMR pathway and excision repair of alkylated bases (N7-MeG and N3-MeA) conducted by the BER pathway. We report the results of a study evaluating the correlation between MGMT, MMR and BER protein expression in tumor tissue of patients (pts) with metastatic melanoma and documented clinical response to chemotherapy. Methods: Frozen tumor tissue was obtained from 17 pts with metastatic melanoma treated with DTIC/TMZ. Tumors were classified as sensitive (S) if pts showed stable disease (SD), partial response (PR) or complete response (CR) by RECIST criteria; and resistant (R) if progressive disease (PD) was observed after 2 cycles of treatment with DTIC/TMZ. Immunohistochemical staining for MGMT, MLH1 (MMR), and DNA polymerase-ß (pol-ß, BER) was performed. Levels of expression were assessed by a pathologist blinded to response data. Fisher’s Exact and Wilcoxon Rank Sum statistical tests were used to assess correlations. Results: 9 tumors were considered to be sensitive (S) and 7 resistant (R). Clinical response was found to significantly correlate with functional MMR (increased MLH1 expression): 7 of 9 S tumors had high MLH1 expression as compared to 1 of 7 R (p-value=0.015). Clinical response was also found to be inversely correlated with pol-ß expression: 6 of 9 S tumors expressed low levels of pol-ß as compared to 1 of 7 R tumors (p-value=0.08). MGMT expression did not seem to correlate with response. Conclusions: The presence of a functional MMR pathway is a requirement for the cytotoxic effect of alkylating agents and was significantly correlated with clinical response. Decreased BER potential (pol-ß expression) was correlated with increased sensitivity to alkylator-based chemotherapy. These findings lend additional support to the view that DNA repair status correlates with response to alkylator-based chemotherapy for melanoma. No significant financial relationships to disclose.