Rainer Klocke

Long‐term expression of differentiated functions in hepatocytes cultured in three‐dimensional collagen matrix

Hepatocytes entrapped in collagen gel and cultured in serum‐free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c‐fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin‐stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen‐cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen‐entrapped cells was maintained above the day‐1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen‐cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione‐transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione‐transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP‐α and ‐β, and HNF‐1 and ‐4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate‐synthetase I). J Cell Physiol 177:553–562, 1998.

Induction of apoptosis and G2/M arrest by infection with replication-deficient adenovirus at high multiplicity of infection

Replication-deficient adenoviruses are among the most widely used vectors in gene therapy and are also becoming increasingly popular as analytical tools in basic research. However, significant toxicity of these vectors in vivo has been reported. Here, we show that in an in vitro setting, first generation adenoviruses lead to growth retardation, prolongation of the G2/M phase and induction of apoptosis if applied at a high multiplicity of infection (MOI). These findings were obtained in p53-deficient hepatocytes, derived from knock-out mice (A2 cells) and in several tumor cell lines containing wild-type (wt) or mutant p53. Apoptosis induction was correlated with increased levels of p53 and bax proteins and it was stronger in cells containing wt p53 as compared with cells lacking functional p53. Apoptosis was highly dependent on the MOI used with marked effects starting at an MOI twice as high as needed for 100% gene transfer. Expression of the adenoviral E4 ORF6 gene as well as adenoviral replication were detected in all cell lines infected with first generation adenovirus. Apoptosis could be considerably reduced but not abrogated by UV inactivation of adenovirus, which indicates proapoptotic effects caused by the infection event as well as by residual adenoviral gene expression or adenoviral replication. First generation adenoviruses apparently display proapoptotic activity if used at higher MOIs, which may be of relevance when these vectors are used as analytical or gene therapeutic tools.

Development of pulmonary bronchiolo-alveolar adenocarcinomas in transgenic mice overexpressing murine c-myc and epidermal growth factor in alveolar type II pneumocytes.

Transgenic mouse models were established to study tumorigenesis of bronchiolo-alveolar adenocarcinomas derived from alveolar type II pneumocytes (AT-II cells). Transgenic lines expressing the murine oncogene c-myc under the control of the lung-specific surfactant protein C promoter developed multifocal bronchiolo-alveolar hyperplasias, adenomas and carcinomas respectively, whereas transgenic lines expressing a secretable form of the epidermal growth factor (IgEGF), a structural and functional homologue of transforming growth factor α (TGFα), developed hyperplasias of the alveolar epithelium. Since the oncogenes c-myc and TGFα are frequently overexpressed in human lung bronchiolo-alveolar adenocarcinomas, these mouse lines are useful as models for human lung bronchiolo-alveolar adenocarcinomas. The average life expectancies of hemizygous and homozygous c-myc transgenics were 14.25 months and 9.2 months, respectively, suggesting that a dosage effect of c-myc caused an accelerated bronchiolo-alveolar adenocarcinoma formation. First analyses of double transgenics, hemizygous for both c-myc and IgEGF, show that these mice develop bronchiolo-alveolar adenocarcinomas at the average age of 9 months, indicating that these oncogenes cooperate during the lung cancer formation. Our results demonstrate that c-myc and EGF are directly involved and cooperate with one another during formation of bronchiolo-alveolar adenocarcinomas in the lung.

Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type II cells

PurposeAs previously described, SPC/myc transgenic mice developed bronchioloalveolar adenocarcinomas derived from alveolar type II (AT II) cells within 10–14xa0months, whereas SPC/IgEGF transgenic mice developed hyperplasias. Our purpose was to determine the potential interplay of environmental and genetic factors in lung tumorigenesis.Materials and methodsSix-week-old SPC/myc and SPC/IgEGF transgenic mice, overexpressing c-myc and a secretable form of the epidermal growth factor (IgEGF) under the control of the surfactant protein C (SPC) promoter, were treated with a single dose of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As control groups, SPC/myc and SPC/IgEGF transgenic mice were treated with NaCl and non-transgenic littermates were treated with NNK or NaCl, respectively.ResultsAfter 6xa0months, none of the NaCl-treated transgenic littermates showed bronchioloalveolar hyperplasia and adenocarcinoma formation, whereas 100% of the NNK-treated SPC/myc transgenic mice did. The effect of NNK on SPC/IgEGF transgenic mice was less pronounced, inducing hyperplasia in the lung in only 16.7% of them. In 90% of the NNK-treated non-transgenic littermates no neoplastic changes were detected in the lung.ConclusionsThese results demonstrate that the progression of pulmonary bronchioloalveolar adenocarcinomas, induced by expression of c-myc as a transgene, was accelerated by NNK, suggesting that c-myc cooperates with NNK-induced mutations.

Uteroglobin promoter-targeted c-MYC expression in transgenic mice cause hyperplasia of Clara cells and malignant transformation of T-lymphoblasts and tubular epithelial cells.

To investigate the influence of the proto-oncogene c-MYC on tumor development in different epithelial tissues which secrete Clara Cell Secretory Protein (uteroglobin, UG), transgenic mouse lines were established expressing the human c-MYC proto-oncogene under the control of the rabbit UG-promoter. These mice expressed the c-MYC transgene in Clara cells and other UG expressing tissues like uterus and prostate. In the bronchioalveolar epithelium of the lung hyperplasias developed originating from Clara cells. Surprisingly, transgenics most frequently developed T-lymphoblastic lymphomas, a polycystic kidney phenotype and renal cell carcinoma derived from tubular epithelial cells, which are both tissues that had so far not been known to express UG. Immunohistological studies in UG/MYC transgenics and in a transgenic line (UG/eGFP) expressing Green Fluorescent Protein confirmed that the uteroglobin promoter is not only active in Clara cells, but also in tubular epithelial cells of the kidney and in lymphatic tissue. The UG/MYC transgenics will be useful to investigate the biochemical mechanisms underlying the development of carcinomas and the oncogenic properties of c-MYC in epithelial cells of various tissues.

Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines

The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

Lack of p53 accelerates hepatocarcinogenesis in transgenic mice constitutively overexpressing c-myc in the liver

The role of the tumor suppressor function of p53 in the process of hepatocellular carcinoma (HCC) development is still enigmatic in view of uncertainties with respect to the phenotype of most p53 mutations found in human HCCs. We therefore analyzed the effect of the p53 knockout (p53KO) genotype, which imparts a clear‐cut loss of function, on hepatocarcinogenesis in livers of transgenic mice prone to develop this tumor type. Deterministic HCC formation in mice overexpressing transgenes encoding murine c‐myc, or c‐myc plus the secretable human epidermal growth factor (EGF) analogue IgEGF, in the liver was accelerated in the p53KO background as indicated by enhanced relative liver weights and reduced survival times of mice. In contrast, HCC formation was not affected by lack of p53 in IgEGF transgenics. Because p53KO mice are not HCC prone, lack of p53 contributed to hepatocarcinogenesis during progression provided that c‐myc was overexpressed in the liver. Thus, deterministic HCC development occurs not simply because of the accumulation of genetic changes in individual hepatocytes; the order in which these changes occur also determines the propensity for liver neoplasia. Enhanced proliferation of hepatocytes was accompanied by markedly reduced p21 levels in tumorous livers of c‐myc/IgEGF/p53KO mice. Concomitantly with unaltered p53‐ independent apoptotic activities, it caused enhanced hyperplasia and accelerated HCC development in livers enlarged fivefold, which killed the mice at 1.9 months of age.

Liver-specific physiology of immortal, functionally differentiated hepatocytes and of deficient hepatocyte-like variants

SummaryFive different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202), consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/ enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the thre cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout, hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.

Constitutive and inducible expression of CYP enzymes in immortal hepatocytes derived from SV40 transgenic mice.

1. The expression of liver-specific transcription factors and cytochrome P450 (CYP) enzymes have been studied in three new hepatocyte-like cell lines derived from SV ▵ 202 transgenic mice: AMH- ▵ 202 (adult mouse hepatocytes), TAMH- ▵ 202 (tumour-derived adult mouse hepatocytes) and NMH- ▵ 202 (newborn mouse hepatocytes). 2. mRNA levels of liver-enriched transcription factors such as D-element binding protein (DBP), liver-enriched transcription activating protein (LAP) and the hepatic nuclear factors (HNF) 1, 2 and 3 in all ▵ 202 transgenic hepatocyte lines were similar to those in the wild-type liver and in primary mouse hepatocytes. 3. Analysis of basal CYP activities and testosterone metabolism revealed that ▵ 202 cells showed higher similarities to mouse hepatocytes than Hepa 1c1c7 hepatoma cells. All three ▵ 202 cell lines exhibited substantial active CYP1A1/2, CYP2A4/5 and CYP3A11 activities and lower levels of CYP2B, CYP2C and CYP2E1 activities. 4. The ▵ 202 cells also responded to model inducers. 3-Methylcholanthrene induced CYP1A1/2 (7-ethoxyresorufin O- deethylation); phenobarbital induced CYP2B (7-benzoxyresorufin O- debenzylation), CYP2A4/5 (testosterone 7 α -hydroxylation) and CYP3A11 (testosterone 6 β -hydroxylation); and rifampicin and dexamethasone induced CYP3A11 activities in the three ▵ 202 cell lines, whereas only AMH- ▵ 202 cells reproduced to a limited extent the response of CYP2E1 to ethanol observed in hepatocytes. 5. The results suggest that generation of hepatocyte lines from transgenic animals constitutes a successful approach to obtain in vitro models alternative to primary hepatocytes for drug metabolism and CYP inducibility studies.

Generation of functional endothelial-like cells from adult mouse germline-derived pluripotent stem cells.

Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells. Cells displaying an endothelial cell-like morphology were obtained from gPS cell-derived embryoid bodies using a combination of fluorescence-activated cell sorting (FACS)-based selection of CD31-positive cells and their subsequent cultivation on OP9 stromal cells in the presence of VEGF-A. Real-time reverse transcriptase polymerase chain reaction, FACS analysis and immunofluorescence staining showed that the gPS cell-derived endothelial-like cells (gPS-ECs) expressed endothelial cell-specific markers including von Willebrand Factor, Tie2, VEGFR2/Flk1, intercellular adhesion molecule 2 and vascular endothelial-cadherin. The high expression of ephrin B2, as compared to Eph B4 and VEGFR3, suggests an arterial rather than a venous or lymphatic differentiation. Their capability to take up Dil-conjugated acetylated low-density lipoprotein and to form capillary-like networks on matrigel confirmed their functionality. We conclude that gPS cells could be a novel source of endothelial cells potentially suitable for regenerative cell-based therapies for ischemic diseases.