Hubert Stöppler

The Human Papillomavirus Type 16 E6 and E7 Oncoproteins Dissociate Cellular Telomerase Activity from the Maintenance of Telomere Length

The “high risk” subgroup of human papillomaviruses (e.g. HPV-16 and HPV-18) infect and induce tumors of mucosal epithelium. These neoplasms, which can progress to malignancy, retain and express the papillomavirus E6 andE7 oncogenes. In vitro, the E6 and E7 proteins associate with the cellular p53 and Rb proteins and interfere with their normal growth-regulatory functions. We report here that primary human keratinocytes transduced with the HPV-16 E6 gene, but not the E7 gene, express significant telomerase activity. However, despite this detectable enzymatic activity,E6-transduced cells continue to shorten their telomeres during in vitro passaging similar to control cells and to cells expressing the E7 and E6+E7 genes. At late passages, however, E7-transduced cells partially restore telomere length, although they lack detectable telomerase activity, demonstrating that E6-independent, telomerase-independent events mediate this change.

Green tea polyphenols reduce autoimmune symptoms in a murine model for human Sjogren's syndrome and protect human salivary acinar cells from TNF-a-induced cytotoxicity

Sjogrens syndrome (SS) is a relatively common autoimmune disorder. A key feature of SS is lymphocytic infiltration of the salivary and lacrimal glands, associated with the destruction of secretory functions of these glands. Current treatment of SS targets the symptoms but is unable to reduce or prevent the damage to the glands. We reported previously that the major green tea polyphenol (GTP) epigallocatechin-3-gallate (EGCG) inhibits autoantigen expression in normal human keratinocytes and immortalized normal human salivary acinar cells (Hsu et al. 2005). However, it is not known whether GTPs have this effect in vivo, if they can reduce lymphocytic infiltration, or protect salivary acinar cells from tumor necrosis factor-α (TNF-α)-induced cytotoxicity. Here, we demonstrate that in the NOD mouse, a model for human SS, oral administration of green tea extract reduced the serum total autoantibody levels and the autoimmune-induced lymphocytic infiltration of the submandibular glands. Further, we show that EGCG protected normal human salivary acinar cells from TNF-α-induced cytotoxicity. This protection was associated with specific phosphorylation of p38 MAPK, and inhibitors of the p38 MAPK pathway blocked the protective effect. In conclusion, GTPs may provide a degree of protection against autoimmune-induced tissue damage in SS, mediated in part through activation of MAPK elements.

Monitoring daunorubicin-induced alterations in protein expression in pancreas carcinoma cells by two-dimensional gel electrophoresis

Tumors of the pancreas are characterized by a high intrinsic potency to develop chemoresistance towards cytotoxic drugs, which is the main cause of ineffective treatment. The phenomenon of multidrug resistance is known to be a multifactorial event in which several mechanisms act simultaneously. We investigated the response of pancreas tumor cells after exposure to the anthracycline daunorubicin (DRC), a well‐known antitumor agent in chemotherapy, by two‐dimensional gel electrophoresis (2‐DE). DRC is known to cause DNA damage and to affect tumor cell growth. Importantly, we aimed at investigating alterations in the protein expression pattern after first contact of the tumor cells with DRC, thus simulating a situation close to clinical chemotherapy and elucidating cell survival strategies following initial drug exposure. A concentration dependent up‐regulation of a variety of proteins was observed, indicating that cell response to DRC involves multiple signaling events. Since the p53 tumor suppressor is essentially involved in the regulation of cell growth and controlled cell death (apoptosis) after cellular stress (like DNA damage), we investigated the role of p53 in DRC‐resistant and ‐sensitive pancreas carcinoma cells by measuring p53 transcriptional transactivation activities. No differences in p53 activities were observed in response to DRC treatment in both pancreas cell lines, whereas mamma carcinoma cells (MCF‐7), possessing wild‐type p53, demonstrated the expected increase in p53 transcriptional transactivation activity. Hence, the tested pancreas carcinoma cells harbor a mutant, nonfunctional p53. We additionally analyzed the steady state protein levels of the cyclin dependent kinase inhibitor p21CIP1, which is known to be involved in cell cycle control. Interestingly, p21CIP1 was induced by DRC in sensitive cells in a concentration dependent manner and was highest in resistant cells. In conclusion, our results suggest that the induction of proteins by DRC in pancreas carcinoma cells, as observed by 2‐DE, occurs independently from p53 signaling events, but is probably associated with increased levels of p21CIP1.

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

BackgroundInfection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.ResultsSix replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27).ConclusionThis large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

The human papillomavirus (HPV) 16 E6 oncoprotein leads to an increase in gene expression of the angiogenic switch molecule FGF-BP in non-immortalized human keratinocytes.

Fibroblast growth factor binding protein (FGF-BP) is a secreted protein that binds FGF-1 and FGF-2 and is involved in mobilization and activation of FGFs from the extracellular matrix. FGF-BP overexpression as well as ribozyme-mediated reduction of endogenous FGF-BP revealed that FGF-BP can be rate-limiting for tumor growth and angiogenesis. Recent studies showed that FGF-BP expression is up-regulated during early phases of tumorigenesis, indicating that the role of FGF-BP in angiogenesis is a critical early step in the development and progression of tumors. Human papillomavirus type 16 (HPV 16) is highly associated with the development of anogenital cancers. Here we demonstrate that the stable expression of the E6 oncogene of HPV 16 leads to an activation of the FGF-BP promoter in primary human foreskin keratinocytes (one of the natural host cells of these viruses). This is associated with an increase in the steady state levels of FGF-BP mRNA and FGF-BP protein in cells stably expressing E6. Transient E6 expression revealed that the observed activation of the FGF-BP promoter by the viral oncogene is an early process which is independent from immortalization/transformation events in the cells.

Telomerase activity of Merkel cell carcinomas and Merkel cell carcinoma-derived cell cultures

Abstract Merkel cell carcinomas are rare malignant tumors of the skin, which are predominantly observed in elderly patients (mean age 65–70 years). It is believed but not yet proven that these tumors are derived from the Merkel cells of the epidermis and hair follicles. The Merkel cells themselves probably originate from an asymmetric cell division of basal keratinocytes and the resulting differentiated Merkel cells have presumably, at least in humans, lost their growth potential. The capability of indefinite cell division in germ line cells and in the great majority of malignant tumors as well as an increased growth potential in certain somatic cells (such as basal cells of renewable tissues) is correlated with cellular telomerase activity, which is absent in differentiated somatic cells. In this study the telomerase activity in cryostat sections of frozen Merkel cell tumor biopsies and in in vitro cultivated Merkel cell carcinoma cells was analyzed. We detected telomerase activity in four tumors and three of four cell cultures. These results show that despite their pronounced neuroendocrine differentiation and their occurrence in patients of advanced age, Merkel cell carcinomas possess telomerase activity similar to that of common carcinoma types.

Cell lines containing and expressing the human herpesvirus 6A ts gene are protected from both H- ras and BPV-1 transformation

Human herpesvirus 6A (HHV-6A) strain U1102 was previously shown to contain a 1473 bp transformation suppressor gene (ts) (Araujo et al., 1995). Ts inhibited transformation of NIH3T3 cells by H-ras and transcription of the H-ras and human immunodeficiency type 1 (HIV-1) promoters in transient transfection experiments. In the current study, stable NIH3T3 cell lines expressing ts protein were established by transfection with pRc-ts containing the ts gene under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) and a neomycin selectable marker. Selected cell lines contained approximately one to two copies per cell of intact ts sequences, expressed ts protein and grew at approximately the same rate as parental NIH3T3 cells. These cell lines were protected from H-ras transformation while parental and NIH3T3 cells containing the ts gene cloned in the antisense orientation were not. Expression of the chloramphenicol acetyl transferase (CAT) gene under the control of the EJ-H-ras promoter was also suppressed in the ts cell lines but not when the CAT gene was under the control of the murine osteosarcoma virus LTR or human cytomegalovirus immediate early promoter. When NIH3T3 cell lines expressing ts protein were established by infection with the retrovirus, LNCts, the cells expressed ts protein and were protected from H-ras transformation. Furthermore, bovine papillomavirus type 1 (BPV-1) transformation was also suppressed in cells co-transfected with BPV-1 plus ts and in ts expressing cell lines transfected with BPV-1. The BPV-1 p89 and p2443 promoters were down-regulated in 3T3-ts lines. Because the human papillomavirus type 16 (HPV-16) p97 promoter has similarity to the BPV-1 p89 promoter, the ability of ts to suppress p97 was also tested. Like the H-ras and BPV-1 promoters, HPV-16 p97 was down-regulated in 3T3-ts lines. The data indicate the utility of ts against H-ras, BPV-1 and HPV-16 promoters and their respective oncogenes.

Merkel Cell Carcinomas Possess Telomerase Activity

Merkel cell carcinomas are believed to be derived from the Merkel cells of the epidermis and hair follicles. The histogenesis of the Merkel cells has not yet been fully elucidated, but several studies suggest that they might originate from an asymmetric cell division of basal keratinocytes or epithelial stem cells of the fetal epidermis, and the resulting differentiated Merkel cells have presumably lost their growth potential.

Influence of human papillomavirus type 16 gene expression on in vitro differentiation of the human teratocarcinoma cell line 2102Ep

Human papillomaviruses (HPVs) are known to infect human keratinocytes and cause alterations in epithelial differentiation. We showed in this study that expression of the HPV‐16 genome was able to interfere with the in vitro differentiation of a human simple‐epithelial cell type, the 2102Ep teratocarcinoma cell line. Stable HPV‐16 genome‐expressing 2102Ep cell lines were generated, and subsequent alterations in differentiation were analyzed in comparison with parental 2102Ep cells. We found that in 2102Ep cells phorbol ester‐induced differentiation led to changes in the expression of SSEA antigens, whereas in HPV‐transfected cell lines only minor changes were observed.