Cloning and Characterization of the Expression Pattern of a Novel Splice Product MIA (Splice) of Malignant Melanoma-derived Growth-inhibiting Activity.

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Cloning and Characterization of the Expression Pattern of a Novel Splice Product MIA (Splice) of Malignant Melanoma-derived Growth-inhibiting Activity (MIAY CD-RAP) Peter Hau, Petra Wise, Piotr Jachimczak, Ines Tschertner, Ulrich Bogdahn, Rainer Apfel Journal of Investigative Dermatology Volume 119, Issue 3, Pages 562-569 (September 2002) DOI: 10.1046/j.1523-1747.2002.00501.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of MIA(splice) in a northern blot analysis. The bands show MIA (runs at 539 bases), MIA(splice) (runs 134 bases lower at 405 bases), and β-actin (used as a control). In the lanes, two cell lines of cerebral metastases of human malignant melanoma, HTZ-19 and CRL-1424 (ATCC), and FV, a human fibroblast cell line, are shown. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western blot analysis of supernatants from cell cultures, showing immunoreactivity to the originally described full length MIA, using a N-terminal specific antibody, as well as to a protein band migrating at the predicted molecular weight of the splice form of about 3.5 kDa. Staining of this lower band is not detectable when using a C-terminal-specific antibody against MIA. In the bands, MIA and MIA(splice) are displayed. In the lanes, a marker, which was enhanced in this figure, and the reaction with a N-terminal antibody in the left panel and a C-terminal antibody in the right panel are shown. In each panel, the lanes show 1 melanoma cell line obtained from ATCC (CRL-1424), two cell lines of cerebral metastasis of human malignant melanoma and two cell lines of human malignant glioma (HTZ-cell lines, established in our laboratory). Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Using a PCR approach, sequencing of MIA(splice) was performed revealing a sequence identical to that of MIA, however, lacking 134 bp of exon 2 of the original sequence and thus resulting in a frameshift. Regular typed amino acids indicate the identical sequence of the proteins MIA and MIA(splice). Italic typed amino acids indicate the unique sequence of MIA and regular and lighter typed amino acids indicate the unique C-terminal protein sequence of MIA(splice). Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The genomic DNA consists of four exons and two noncoding introns. Exon 1 consists of the bases for the first 18 amino acids of the peptide as described for MIA above. After complete lack of exon 2, a frameshift causes a different amino-acid sequence in the second part of the peptide. An additional Histidine residue (amino acid 43) is generated by regular splicing of exon 1 to exon 2 and is not indicated here. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reverse transcriptase–PCR from tissue of six naevi, six melanomas, 12 primary sites of metastatic melanomas, 10 systemic metastasis of melanomas, four CNS metastasis of melanomas, eight primary melanoma cultures, and eight controls (basalioma, n = 4; endothelium, n = 1; healthy colon, n = 1; colitis, n = 1; and carcinoma of the colon, n = 1). In the lanes, MIA(splice), MIA, and GAPDH (control) are shown for each specimen; marker 8 (Roche Pharma, Roche Diagnostics AG, Industriestrasse 7, 6343 Rotkreuz, Switzerland) was used as a weight marker. Expression levels of melanocytic tumors correlate with the malignancy of tumors. MIA(splice) can be detected in nonmelanocytic controls as well, but to a much weaker extent. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 PAP stain of different tumors and fibroblasts treated with serum of a rabbit immunized against MIA(splice) (A1, first row, left) and MIA (B1, C1, D1, E1, F1, next rows, left), and controls (A2, B2, C2, D2, E2, F2, right). (A,B) HTZ-19, malignant melanoma; (C) HTZ-318, malignant melanoma; (D) HTZ-17, glioblastoma; E: HTZ-146, glioblastoma; and (F) H-36, embryonal fibroblasts. With the antibody generated against MIA(splice), a more nuclear staining pattern is detected in comparison with the antibody against MIA, where a more cytoplasmatic staining pattern was detected. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 PAP stain of different stages of melanocytic tumors treated with the serum of a rabbit immunized against MIA(splice). (A) Naevus; (B) primary site of malignant melanoma; (C) metastasis of malignant melanoma. A very faint nuclear staining pattern is detected in the nevus, whereas in the primary site melanoma, staining is enhanced. In the metastasis, the most prominent nuclear staining pattern was detected. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Northern blot of the melanoma cell line HTZ-19 treated with TGF-β2 10 ng per ml. Expression of MIA and MIA(splice) was analyzed by harvesting cells after 3, 6, 12, 24, and 48 h and isolating mRNA. The bands show MIA and MIA(splice), the lanes show labeled mRNA at the given time points and a untreated control. After incubation with TGF-β2, MIA is downregulated, whereas MIA(splice) is upregulated. At 48 h, both proteins are back to normal levels. Journal of Investigative Dermatology 2002 119, 562-569DOI: (10.1046/j.1523-1747.2002.00501.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions