Repression of genes involved in melanocyte differentiation in uveal melanoma.

PURPOSE: Uveal melanoma (UM) has been the subject of intense interest due to its distinctive metastatic pattern, which involves hematogenous dissemination of cancerous cells toward the liver in 50% of patients. To search for new UM prognostic markers, the Suppressive Subtractive Hybridization (SSH) technique was used to isolate genes that are differentially expressed between UM primary tumors and normal uveal melanocytes (UVM). METHODS: A subtracted cDNA library was prepared using cDNA from uncultured UM primary tumors and UVM. The expression level of selected genes was further validated by cDNA microarray, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence analyses. RESULTS: One hundred-fifteen genes were identified using the SSH technique. Microarray analyses comparing the gene expression profiles of UM primary tumors to UVM validated a significant differential expression for 48% of these genes. The expression pattern of selected genes was then analyzed by semi-quantitative RT-PCR and was found to be consistent with the SSH and cDNA microarray findings. A down-regulation of genes associated with melanocyte differentiation was confirmed in UM primary tumors. Presence of undifferentiated cells in the UM was demonstrated by the expression of stem cell markers ATP-binding cassette sub-family G member 2 (ABCG2) and octamer-binding protein 4 (OCT4). CONCLUSIONS: We demonstrated that the SSH technique is efficient to detect differentially expressed genes between UM and UVM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor. In addition, deregulation of the melanocyte differentiation pathway revealed the presence of UM cells exhibiting a stem cell-like phenotype.

Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization.

PURPOSE: Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique. METHODS: The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT-PCR and western blot analyses. RESULTS: Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT-PCR and western blot, and was found to be consistent with the SSH findings. CONCLUSIONS: We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor.

Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

Control of integrin genes expression in the eye.

The ending race for sequencing the human genome has left the scientists faced with new challenges. Indeed, now that almost every human gene has been sequenced and precisely positioned on the human chromosomes, one of the next, most burning task consist in understanding how transcription of these genes is ensured in any given cell. The integrins encoding genes are no exception. Integrins bridge the cell to the many components from the extracellular matrix (ECM), such as laminins (LM) and collagens, and thereby transduce intracellular signals that will alter many of the cell's properties such as adhesion, migration, proliferation and survival. As a much clearer picture of the many proteins that belong to this family has emerged over the last few years, tremendous efforts have been dedicated to the identification of the regulatory sequences that modulate their expression. This review provides an overview of the current state of knowledge about the organization of the regulatory elements and the transcription factors (TF) they bind that are used by the cell in order to ensure transcription of each of the integrins gene. A particular attention has been given to those reported to be expressed in the eye. It also explores how components from the ECM might participate in the control of integrins gene expression and establishes links to wound healing of the corneal epithelium, a process that transiently alter the composition of the basement membrane on which the epithelial cells lie.

Laminin reduces expression of the human alpha6 integrin subunit gene by altering the level of the transcription factors Sp1 and Sp3.

PURPOSE: Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after corneal injury, which is later replaced by the secretion of laminin (LM). Laminin is recognized by receptors (alpha6beta1 and alpha6beta4) that belong to the integrin family. The authors characterized the regulatory influence exerted by laminin on the Sp1/Sp3-mediated alpha6 gene promoter function. METHODS: Recombinant plasmids bearing the CAT reporter gene, fused to various segments from the alpha6 promoter, were transfected into rabbit corneal epithelial cells (RCECs) grown on untreated or on LM-coated culture plates or into Sp1-deficient Drosophila Schneider cells. Expression and DNA binding of Sp1/Sp3 was monitored with the use of Western blot and electrophoretic mobility shift assays (EMSAs), respectively. DNA target sites for Sp1/Sp3 in the alpha6 gene promoter were identified in vitro by DNAse I footprinting. Binding of Sp1 and of a few other transcription factors was also examined in vivo by chromatin immunoprecipitation (ChIP) assays. Expression of the alpha6 mRNA in RCECs grown on LM-coated culture plates was also assessed by Northern blot and RT-PCR analyses. RESULTS: Transfection experiments provided evidence that both Sp1 and Sp3 positively influence alpha6 promoter activity in Sp1/Sp3-deficient SL2 Schneider cells. Two GC-rich target sites for Sp1/Sp3 (a proximal and a distal site) were identified in the alpha6 promoter by DNAse I footprinting. Binding of Sp1/Sp3 was further validated in vivo by ChIP. Transfections conducted into RCECs grown on LM-coated culture plates resulted in repression of the activity directed by the alpha6 promoter. This LM-mediated negative regulatory influence also translated into a similar reduction in the expression of the endogenous alpha6 mRNA as revealed by both RT-PCR and Northern blot analyses. Most of all, results from EMSA and Western blot analyses suggested that the LM-mediated repression of the alpha6 promoter activity results in part from the proteolytic cleavage of Sp1/Sp3. CONCLUSIONS: Unlike FN, which functions as an activator of alpha5 gene transcription, LM repressed transcription, directed by the alpha6 gene promoter, by altering the nuclear levels of Sp1 and Sp3. Reappearance of LM in the basement membrane after repair of the corneal damage is therefore expected to substantially contribute to the final steps of this process by influencing the degree to which genes that encode protein products requested for cell adhesion and migration, such as integrin genes, are expressed during corneal wound healing.

Influence of sp1/sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues.

PURPOSE: Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS: HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS: Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS: Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.

Qualitatively monitoring binding and expression of the transcription factor Sp1 as a useful tool to evaluate the reliability of primary cultured epithelial stem cells in tissue reconstruction.

Electrophoretic mobility shift assay and Western blot are simple, efficient, and rapid methods for the study of DNA-protein interactions and expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem-cells through the culture process is essential to produce high quality substitutes. However as such cells are passaged in culture, they often lose their ability to proliferate, a process likely to be determined by the altered expression of nuclear-located transcription factors such as Sp1, whose expression has been documented to be required for cell adhesion, migration, and differentiation. Our recent studies demonstrated that reconstructed tissues exhibiting poor histological and structural characteristics are also those that were produced with epithelial cells in which expression and DNA binding of Sp1 was reduced in vitro. Therefore, monitoring both the expression and DNA binding of this transcription factor in human skin and corneal epithelial cells might prove a particularly useful tool for selecting which cells are to be used for tissue reconstruction.

Expression of the α5 integrin gene in corneal epithelial cells cultured on tissue-engineered human extracellular matrices.

The integrin α5ß1 plays a major role in corneal wound healing by promoting epithelial cell adhesion and migration over the fibronectin matrix secreted as a cellular response to corneal damage. Expression of α5 is induced when rabbit corneal epithelial cells (RCECs) are grown in the presence of fibronectin. Here, we examined whether α5 expression is similarly altered when RCECs or human corneal epithelial cells (HCECs) are grown on a reconstructed stromal matrix used as an underlying biomaterial. Mass spectrometry and immunofluorescence analyses revealed that the biomaterial matrix produced by culturing human corneal fibroblasts with ascorbic acid (ECM/35d) contains several types of collagens, fibronectin, tenascin and proteoglycans. Results from transfection of CAT/α5-promoter plasmids, Western blot and EMSA analyses indicated that ECM/35d significantly increase expression of α5 in HCECs as a result of alteration in the expression and DNA binding of the transcription factors NFI, Sp1, AP-1 and PAX6. The biological significance of this biomaterial substitute on the expression of the α5 gene may therefore contribute to better understand the function played by the α5ß1 integrin during corneal wound healing.

Rescue of the transcription factors Sp1 and NFI in human skin keratinocytes through a feeder-layer-dependent suppression of the proteasome activity.

Co-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.

Altered expression of the poly(ADP-ribosyl)ation enzymes in uveal melanoma and regulation of PARG gene expression by the transcription factor ERM.

PURPOSE: Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties. METHODS: Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice. RESULTS: Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation. CONCLUSIONS: Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo.

Suppression of α5 gene expression is closely related to the tumorigenic properties of uveal melanoma cell lines.

Cancer aggressiveness is related to the ability of cancer cells to escape the anchorage dependency toward the extracellular matrix, a process regulated by the integrin α5ß1 and its ligand fibronectin. Here, we characterized the expression of the α5 gene in human uveal melanoma cell lines with distinct tumorigenic properties and investigated some of the mechanisms underlying the variations of their malignancy. Strong and weak expression of α5 was observed in cells with no (T108/T115) and high (T97/T98) tumorigenic properties, respectively. Expression and DNA binding of the transcription factors Sp1, activator protein 1 (AP-1) (both acting as activators), and nuclear factor I (NFI) (a strong repressor) to the α5 promoter were demonstrated in all cell lines. A reduced expression of AP-1 combined with a dramatic increase in NFI correlated with the suppression of α5 expression in T97 and T98 cells. Restoring α5 expression in T97 cells entirely abolished their tumorigenicity in immunodeficient mice. These uveal melanoma cell lines might therefore prove particularly useful as cellular models to investigate α5ß1 function in the pathogenesis of invasive uveal melanoma.

Tissue engineering of skin and cornea: Development of new models for in vitro studies.

Human beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donor's age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age-dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue-engineered skin as a model, we linked Hsp27 activation with skin differentiation.

Transcriptional regulation of the human alpha6 integrin gene by the transcription factor NFI during corneal wound healing.

PURPOSE: Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human alpha6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human alpha6 gene, and its regulatory influence was characterized in corneal epithelial cells. METHODS: Plasmids bearing the alpha6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the alpha6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. RESULTS: All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of alpha6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of alpha6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. CONCLUSIONS: Repression of alpha6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane.