Linda K. Hansen

Evidence That Cyclin D1 Mediates Both Growth and Proliferation Downstream of TOR in Hepatocytes

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regenerationin vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers.

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via β1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH: OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the β1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the β1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. ɛ-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly.

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.

Differential regulation of HSC70, HSP70, HSP90α, and HSP90β mRNA expression by mitogen activation and heat shock in human lymphocytes☆

Abstract A subset of heat shock proteins, HSP90α, HSP90β, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90α appears more inducible by heat shock than mitogens while the opposite is true for HSP90β. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.

Three-dimensional co-culture of hepatocytes and stellate cells.

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.

Hepatocyte adhesion, growth and differentiated function on RGD-containing proteins.

There is currently much interest in designing synthetic substrates incorporating the cell binding motif RGD for tissue engineering. In this paper, hepatocyte function was examined on two synthetic RGD substrates and compared to that on fibronectin (Fn). One is a 2.3 kD RGD peptide (P-2) containing a single RGD, a short spacer in the middle and an end basic sequence to enhance adsorption. On bacteriological plastic, P-2 induced a rounded cell shape, enhanced differentiated function, and inhibited DNA synthesis. The other, a 73 kD synthetic RGD protein Pronectin F (PnF), contains repeating RGD units interspersed with a structural peptide. PnF induced cell spreading, dedifferentiation, and enhanced DNA synthesis, similar to Fn. In addition, only P-2 showed distinct differences in cell shape and DNA synthesis when coated on bacteriological plastic, or on Immulon II plastic, both intrinsically non-adhesive to cells. On bacteriologic plates coated with P-2, cells were round and showed diminished DNA synthesis while on Immulon II plates, they were spread and showed enhanced DNA synthesis. These results demonstrate that synthetic RGD peptides can induce very different hepatocyte function depending on the context in which they are presented to cells. It is likely that the RGD peptide conformation determines the specificity of cellular response.

Regulation of hepatocyte cell cycle progression and differentiation by type I collagen structure.

Cell behavior is strongly influenced by the extracellular matrix (ECM) to which cells adhere. Both chemical determinants within ECM molecules and mechanical properties of the ECM network regulate cellular response, including proliferation, differentiation, and apoptosis. Type I collagen is the most abundant ECM protein in the body with a complex structure that can be altered in vivo by proteolysis, cross-linking, and other processes. Because of collagens complex and dynamic nature, it is important to define the changes in cell response to different collagen structures and its underlying mechanisms. This chapter reviews current knowledge of potential mechanisms by which type I collagen affects cell behavior, and it presents data that elucidate specific intracellular signaling pathways by which changes in type I collagen structure differentially regulate hepatocyte cell cycle progression and differentiation. A network of polymerized fibrillar type I collagen (collagen gel) induces a highly differentiated but growth-arrested phenotype in primary hepatocytes, whereas a film of monomeric collagen adsorbed to a rigid dish promotes cell cycle progression and dedifferentiation. Studies presented here demonstrate that protein kinase A (PKA) activity is significantly elevated in hepatocytes on type I collagen gel relative to collagen film, and inhibition of this elevated PKA activity can promote hepatocyte cell cycle progression on collagen gel. Additional studies are presented that examine changes in hepatocyte cell cycle progression and differentiation in response to increased rigidity of polymerized collagen gel by fiber cross-linking. Potential mechanisms underlying these cellular responses and their implications are discussed.

Induction of hepatocyte proliferation and liver hyperplasia by the targeted expression of cyclin E and skp2

Cells in culture become competent to replicate in the absence of growth factor after progressing beyond the late G1 restriction point, suggesting that a set of genes expressed during G1 phase is sufficient to trigger completion of the cell cycle. However, this has not been demonstrated in an in vivo system. In this study, we examined whether transfection of genes associated with the G1/S transition could trigger hepatocyte replication. Co-transfection of cyclin E and skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the absence of mitogen or in the presence of growth inhibitors. Furthermore, transfection of hepatocytes in vivo with cyclin E and skp2 promoted abundant hepatocyte replication and hyperplasia of the liver. These studies confirm that transfection with a small number of genes can trigger proliferation of quiescent hepatocytes in vivo, and suggest that therapies to enhance liver regeneration by targeting cell cycle control genes may be feasible.

Cutaneous-delayed hypersensitivity in nursing home and geriatric clinic patients. Implications for the tuberculin test.

Cutaneous‐delayed hypersensitivity was studied by one and two‐step Mantoux‐type skin tests to four standard antigens in 33 elderly nursing home residents, 34 geriatric clinic patients, and 20 healthy young adult controls. Demographic and anthropometric data were collected to determine the effects of nutrition and other variables on cutaneous‐delayed hypersensitivity. Anergy (a lack of response greater than 5 mm of induration when read at 48 hours) to any of the four antigens occurred in 34% of nursing home residents, 17% of geriatric clinic patients, and none of the healthy young adults. Mean and maximal responses were less in the nursing home residents than the clinic patients or controls, even if anergic individuals were excluded from analysis, suggesting both a qualitative and quantitative decline in cell‐mediated immune function in this elderly population. Repeat testing with each antigen for which there was a negative initial response revealed a “booster” affect of 7 to 19% and occurred as commonly in the healthy young adults as in the nursing home residents or geriatric clinic patients. The mumps antigen elicited strong responses in the healthy young adults, but weak reactions in the nursing home residents. An unexpectedly high prevalence of positive tuberculin (PPD) responses occurred in the nursing home residents, suggesting recent exposure. Analysis of anthropometric and demographic characteristics show that neither nutritional status nor age alone can account for differences in cutaneous‐delayed hypersensitivity observed between populations. Cutaneous‐delayed hypersensitivity may vary widely between elderly populations and have important practical implications for the tuberculin test.

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer

Tissue-like structures of cells organized in vitrohave a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.