Biomedical subjects

Thomas Eichberger

Publications and source records attributed to Thomas Eichberger.

7 recordsLinked to original sources

PubMed2006

RNA expression profiling at the single molecule level.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Cell Line↗

PubMed2006

Overlapping and distinct transcriptional regulator properties of the GLI1 and GLI2 oncogenes.

The GLI transcription factors mediate the hedgehog signal in development and carcinogenesis. Basal cell carcinoma can be caused by overexpression of either GLI1 or GLI2. Though GLI1 and GLI2 have identical or very similar DNA binding specificities, some of their activities are overlapping, some are clearly distinct. We analyzed target gene specificities of GLI1 and constitutively active GLI2 (GLI2DeltaN) by global expression profiling in an inducible, well-characterized HaCaT keratinocyte expression system. Four hundred fifty-six genes up- or downregulated at least twofold were identified. GLI target gene profiles correlated well with the biological activities of these transcription factors in hair follicles and basal cell carcinoma. Upregulation of largely overlapping sets of target genes was effected by both factors, repression occurred predominantly in response to GLI2. Also, significant quantitative differences in response to GLI1 and GLI2DeltaN were found for a small number of activated genes. Since we have not detected a putative processed GLI2 repressor, these results point to specific but indirect target gene repression by GLI2DeltaN via preferential activation of one or more negative regulators.

Animals↗

PubMed2004

Activation of the BCL2 promoter in response to Hedgehog/GLI signal transduction is predominantly mediated by GLI2.

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Base Sequence↗

PubMed2004

The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells.

In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1-S phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

Base Sequence↗

PubMed2004

FOXE1, a new transcriptional target of GLI2 is expressed in human epidermis and basal cell carcinoma.

Sonic hedgehog (Hh) signaling plays a key role in epidermal development and skin cancer. Mutational inactivation of the tumor suppressor gene patched (PTCH) leads to constitutive activation of the Hh signaling pathway, resulting in activation of target gene transcription by the zinc finger transcription factors GLI1 and GLI2. Recent experiments in mice point to GLI2 as the key mediator of Hh signaling in skin. We have concentrated on the identification of candidate mediators of GLI2 function in the human epidermis. We show here that the forkhead/winged-helix domain transcription factor FOXE1 is likely to be a direct GLI2 target gene. The kinetics of FOXE1 induction are similar to the known direct target PTCH, and a 2.5 kb upstream fragment containing five GLI-binding sites activates transcription in a reporter assay. We show by in situ hybridization that FOXE1 is expressed in the outer root sheath of the hair follicle, where murine Gli2 is also expressed. FOXE1 expression is also found in basal keratinocytes of the human epidermis and basal cell carcinoma (BCC). These data point to a putative role of FOXE1 in mediating Hh signaling in the human epidermis downstream of GLI2.

Basal Cell Carcinoma↗

PubMed2004

GLI2 is expressed in normal human epidermis and BCC and induces GLI1 expression by binding to its promoter.

Sonic hedgehog (Shh) binds to its receptor patched (PTCH), leading to the activation and repression of target genes via the GLI family of zinc-finger transcription factors. Deregulation of the Shh pathway is associated with basal cell carcinoma (BCC) due to upregulation of GLI1 and GLI2. We recently demonstrated a positive feedback loop between GLI1 and GLI2, which revealed that GLI1 may be a direct target of GLI2. Using band shift and luciferase reporter assays, we now show that GLI2 binds the GLI-binding consensus sequence in the GLI1 promoter. These data suggest that GLI2 directly activates GLI1 and that retrovirally expressed GLI2 induces expression of endogenous GLI1 in human primary keratinocytes. Finally, using in situ hybridization, we show that GLI2 is expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumor islands. These results suggest an important role for GLI2 in regulating epidermal proliferation and skin tumorigenesis.

Base Sequence↗

PubMed2002

Human GLI2 and GLI1 are part of a positive feedback mechanism in Basal Cell Carcinoma.

Transgenic mouse models have provided evidence that activation of the zinc-finger transcription factor GLI1 by Hedgehog (Hh)-signalling is a key step in the initiation of the tumorigenic programme leading to Basal Cell Carcinoma (BCC). However, the downstream events underlying Hh/GLI-induced BCC development are still obscure. Using in vitro model systems to analyse the effect of Hh/GLI-signalling in human keratinocytes, we identified a positive feedback mechanism involving the zinc finger transcription factors GLI1 and GLI2. Expression of GLI1 in human keratinocytes induced the transcriptional activator isoforms GLI2alpha and GLI2beta. Both isoforms were also shown to be expressed at elevated levels in 21 BCCs compared to normal skin. Detailed time course experiments monitoring the transcriptional response of keratinocytes either to GLI1 or to GLI2 suggest that GLI1 is a direct target of GLI2, while activation of GLI2 by GLI1 is likely to be indirect. Furthermore, expression of either GLI2 or GLI1 led to an increase in DNA-synthesis in confluent human keratinocytes. Taken together, these results suggest an important role of the positive GLI1-GLI2 feedback loop in Hh-mediated epidermal cell proliferation.

Alkaline Phosphatase↗