CTGF is overexpressed in malignant melanoma and promotes cell invasion and migration.

BACKGROUND: Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes most likely favour tumour growth and progression, and influence tumour environment. The induction of transforming growth factor beta1, 2 and 3 as well as BMP4 and BMP7 expression in malignant melanoma has been reported before, whereas the expression of an important modulator of these molecules, connective tissue growth factor (CTGF), has not been investigated in melanomas until now. METHODS: Expression of CTGF was analysed in melanoma cell lines and tissue samples by qRT-PCR and immunohistochemistry. To determine the regulation of CTGF expression in malignant melanoma, specific siRNA was used. Additionally, migration, invasion and attachment assays were carried out. RESULTS: We were able to demonstrate that CTGF expression is upregulated in nine melanoma cell lines and in primary and metastatic melanoma in situ. The transcription factor HIF-1α was revealed as a positive regulator for CTGF expression. Melanoma cells, in which CTGF expression is diminished, show a strong reduction of migratory and invasive properties when compared with controls. Further, treatment of normal human epidermal melanocytes with recombinant CTGF leads to an increase of migratory and invasive behaviour of these cells. CONCLUSION: These results suggest that CTGF promotes melanoma cell invasion and migration and, therefore, has an important role in the progression of malignant melanoma.

Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

Induction of AP-2alpha expression by adenoviral infection involves inactivation of the AP-2rep transcriptional corepressor CtBP1.

AP-2 transcription factors execute important functions during embryonic development and malignant transformation. Recently, we have isolated a transcriptional repressor of AP-2alpha expression, the novel Krüppel-related zinc finger protein AP-2rep (Klf12). Here, we show that repression of AP-2alpha transcription by AP-2rep is dependent on an N-terminal PVDLS motif that interacts specifically with the corepressor CtBP1 both in vivo and in vitro. This interaction motif was previously identified in the C-terminal region of the adenoviral oncoprotein E1A. Infection of both HeLa and PA-1 cells with adenovirus type 5 strongly induced AP-2alpha mRNA. Consistently, E1A was necessary and sufficient to mediate up-regulation of AP-2alpha. Transiently transfected wild-type E1A protein activated an AP-2rep sensitive cis-regulatory element of the AP-2alpha promoter, but E1A protein harboring a mutation in the PVDLS motif failed to activate. In summary, we conclude that the adenoviral oncoprotein E1A activates transcription from the endogenous AP-2alpha gene, an effect that involves transcriptional derepression of the AP-2alpha promoter by interaction of E1A with the AP-2rep corepressor CtBP1.

Deficiency of a novel retinoblastoma binding protein 2-homolog is a consistent feature of sporadic human melanoma skin cancer.

Using RNA arbitrarily primed PCR, the authors selected for transcripts with cell cycle-related differential expression in cultured human melanocytes. Among the partial cDNAs cloned, a novel cDNA was identified, which showed 54% identity to the recently cloned cDNA of the retinoblastoma binding protein-2 (RBP2). The 6.5-kB full-length cDNA of this RBP2-related gene, termed RBP2 homolog 1 (RBP2-H1), was obtained from a human teratocarcinoma cDNA library. Two independent libraries from human malignant melanomas were negative. A computerized sequence analysis revealed highly conserved motifs with possible functional meaning: two domains that, in the RBP2 homolog, mediate the binding and interaction with the proteins encoded by the retinoblastoma susceptibility gene, the TATA-binding protein, and the oncoprotein rhombotin 2; in addition, two DNA-binding zinc finger/leukemia-associated protein motifs were detected. Because a functional role in cell-cycle control and transcriptional activation can be envisioned, we investigated the expression of this novel transcript in normal fetal and adult tissues, as well as tissues of benign and malignant melanocytic tumors. By conducting multiple Northern blot, RT-PCR, and in situ hybridization analyses, the authors showed that the corresponding mRNA is expressed in virtually all normal tissues. Accordingly, they found RBP2-H1 expression in microdissected tissue samples from benign melanocytic nevi (n = 10). In contrast, the transcript is significantly down-regulated or even lost in tissue samples from human malignant melanomas (n = 13), melanoma metastases (n = 10), and melanoma cell lines (n = 7). The authors concluded that the loss or down-regulation of RBP2-H1 expression could be a useful molecular marker for a transformed phenotype in the human melanocytic system.

Mouse CD-RAP/MIA gene: structure, chromosomal localization, and expression in cartilage and chondrosarcoma.

A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al. [1994] Cancer Res. 54:5695-5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell [1996] J. Biol. Chem. 271:3311-3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NF kappa B, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor beta 1 (Tgf-beta 1) and the glucose phosphate isomerase 1 (Gpi 1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (XsJ); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma.

Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones.

Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix.