Functional Analysis of the Human Calcyclin Gene Promoter in a Panel of Human Melanoma Cell Lines

Abstract

By comparing two subsequent human tumor stages we previously described calcyclin as a new potential melanoma associated neoplastic progression marker positively linked with metastasis. In this study the calcyclin expression levels in a representative panel of human melanoma cell lines were correlated with the occurrence of DNase I hypersensitive (DH) regions and potential enhancer elements in a 6 kb genomic fragment spanning the human calcyclin gene. Examination of the chromatin structure of the transcription unit revealed no qualitative differences in DH sites within the panel of tested human melanoma cells, but especially the sequences around the transcription start site and a 1.5 kb upstream region appeared more accessible to the nuclease in frequently (BLM, MV3) as compared to poorly (530, 1F6) metastasizing cells. The genomic fragments that harbor one or more DH sites were subjected to functional analysis by luciferase reporter gene assays. Thus, an enhancer element was detected between 361 and 167 bp upstream of the transcription start site. This enhancer displayed equal activating potential (2-3 fold) both in weakly and in frequently metastasizing cells and was apparently recognized by transcription factors present in both types of human melanoma cell lines. We. conclude that, in addition to a slight amplification of the encoding gene, the elevated calcyclin mRNA levels are only reflected in a selectively increased accessibility of the chromatin structure to DNasel in metastasizing melanoma cells © 1995 Academic Preas, Inc.Calcyclin is a small calcium-binding protein (10.5 kD) belonging to the SI00 family containing two calcium-binding structures, the so called EF hands. It was identified as a differentially expressed mRNA in serum-stimulated and quiescent fibro blasts [1,2] and was originally isolated from Gr specific temperature sensitive mutants of baby Syrian hamster kidney cells [3].Although the function of calcyclin is still unknown, several observations can be made: the calcyclin gene was induced by serum, platelet-derived growth factor, or epidermal growth factor [1,4,5]. The gene product was shown to bind both zinc and calcium [6] which indicated that it is involved in processes related to signal transduction.

Protein Expression Profiles in the Epidermis of Cyclooxygenase-2 Transgenic Mice by 2-Dimensional Gel Electrophoresis and Mass Spectrometry

“…S100 proteins belong to a family of 21 low molecular weight calcium binding proteins. − These proteins were implicated in various biological functions in a calcium-dependent manner, such as enzyme activity, protein phosphorylation, calcium homeostasis, cell growth and differentiation. − Of the two S100 proteins identified in the present study, S100A6 was down-regulated 2.5-fold in the epidermis of untreated K14.COX-2 TG compared to that of wild-type control mice. Several previous studies showed that S100A6 expression was increased in many types of cancers. − The higher expression of S100A6 was also suggested to be associated with the extent of colorectal adenocarcinoma invasion/metastasis in yet another study …”

“…Several previous studies showed that S100A6 expression was increased in many types of cancers. [54][55][56] The higher expression of S100A6 was also suggested to be associated with the extent of colorectal adenocarcinoma invasion/metastasis in yet another study. 57 Although the biological function of S100A6 remains unknown, its decreased protein level in epidermis of K14.COX-2 TG, in contrast to its overexpression in many tumors, correlates with the resistance of these mice to TPA tumor promotion.…”

Expression of the PDGF α‐receptor 1.5 kb transcript, OCT‐4, and c‐KIT in human normal and malignant tissues. Implications for the early diagnosis of testicular germ cell tumours and for our understanding of regulatory mechanisms

“…Melanocyte total RNA, isolated from a routinely grown primary culture using the RNeasy mini kit (Qiagen), was kindly provided by Dr N. Smit (Department of Dermatology, Academic Hospital, Leiden, The Netherlands). Total RNA from the Mel 57 and 1F6 cell lines22 was kindly provided by Dr William Leenders. Poly(A+) RNA isolated from U‐1240 MG, U‐251 MGsp, U‐1242 MG, and U‐374 MG malignant glioma cell lines23 was kindly provided by Dr Ester Piek (Ludwig Institute for Cancer Research, Uppsala, Sweden).…”

Binding and functional characteristics of two E‐box motifs within the S100A6 (calcyclin) gene promoter

“…Plasmids pGL2 vectors containing luciferase gene and the À1371/þ134 and À361/þ134 fragments of the human S100A6 gene promoter were described earlier [van Groningen et al, 1995]. Plasmid bearing the À361/þ134 promoter fragment with the E-box sequence at position À283/À278 (Ebox1) mutated to CAGCTT was obtained as described [Leśniak et al, 2000].…”