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RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi  Peter Helmbold, Antje M. Richter, Sara.

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Presentation on theme: "RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi  Peter Helmbold, Antje M. Richter, Sara."— Presentation transcript:

1 RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi  Peter Helmbold, Antje M. Richter, Sara Walesch, Alexander Skorokhod, Wolfgang Ch Marsch, Alexander Enk, Reinhard H. Dammann  Journal of Investigative Dermatology  Volume 132, Issue 3, Pages (March 2012) DOI: /jid Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 RNA expression of RASSF2 and RASSF10 in malignant melanoma (MM) cell lines. MM cell lines (buf1280, C8161, IGR1, and MeWo) were treated for 4 days with the indicated concentrations of 5-aza-deoxycyticine (aza). RNA was isolated and analyzed by reverse transcription PCR (RT-PCR). (a) Products for RASSF2 (236bp) and RASSF10 (243bp) and a 100bp ladder (M) were resolved on 2% gel. Expression of β-actin (ACTB; 225bp) was determined as a control for RNA integrity. (b) Reexpression of RASSF2 was analyzed by quantitative RT-PCR, normalized to ACTB expression, and plotted relative to the C8161 control (=1). (c) Expression of RASSF10 was normalized to ACTB and plotted relative to C8161 (=1). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Methylation analysis of RASSF2. Methylation of the RASSF2 promoter in (a) malignant melanoma (MM) cell lines, (b) nevus cell nevi (NCN), skin metastases of MM (SM), or primary MM (superficial spreading melanoma (SSM)), and (c) brain metastases of MM (BM). The methylation status of the RASSF2 promoter was analyzed by combined bisulfite restriction analysis. PCR products (100bp or 224bp) from bisulfite-treated DNA were digested (TaqI, T) and mock digested (-) and resolved on 2% gel. In vitro-methylated DNA (Methy) was used as positive control. Sizes of a 100bp ladder (M) are marked. Methy, methylation control; m, methylated; pm, partially methylated; u, unmethylated; numbers in brackets, case numbers of Supplementary Tables S1 and S2 online. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Methylation analysis of RASSF10. Methylation of the RASSF10 promoter in (a) malignant melanoma (MM) cell lines, (b) nevus cell nevi (NCN) or primary MM (superficial spreading melanoma (SSM), nodular melanoma (NM)), and (c) MM metastasis. PCR products (241bp) were digested (TaqI, T) and mock digested (-) and resolved on 2% gel. The arrowheads indicate a digest product at 192bp. Sizes of a 100bp ladder (M) are marked (Methy, methylation control; m, methylated; pm, partially methylated; u, unmethylated; numbers in brackets, case numbers of Supplementary Tables S1 and S2 online). BM, brain metastasis; LNM, lymph node metastasis; SM, skin metastasis. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Methylation analysis of the RASSF10 promoter by pyrosequencing. PCR products obtained from bisulfite PCR of malignant melanoma (MM) cell lines (C8161, buf1280, and IGR1), non-Hodgkin lymphoma (NHL, diffuse large B-cell lymphoma of the skin), nodular MM (NM), and dyplastic (Clark) nevus cell nevi (NCN) were analyzed by pyrosequencing. The frequency of CpG methylation is indicated (arrowheads, bisulfite controls; numbers in brackets, case numbers of Supplementary Tables S1 and S2 online). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Comparison between malignant melanoma (primaries, metastases) and nevus cell nevi (including dysplastic Clark nevi). Bar chart indicates the number of tumors with unmethylated or hypermethylated RASSF10 promoter regions. Fisher's exact test. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Microphotographs of selected samples (case numbers ofSupplementary Tables S1 and S2 online); RASSF10 promoter state: M, hypermethylated; U, unmethylated (□). (a) Compound nevus (case 79). (b) Dysplastic (Clark) nevus (case 96). (c) Superficial spreading melanoma (case 18). (d–f) Clark nevus with initial transition into malignant melanoma (MM; case 7). (f) Focal intraepidermal transmigration of melanocytes (melan A immunostaining). (g–k) Anti-RASSF10 antibody (red). (g) Compound nevus with unmethylated RASSF10 promoter, RASSF10 expression in nevus tissue (*), epidermis (arrow), and connective tissue cells. (h) Isotype control. (i) Skin metastasis of MM with hypermethylated RASSF10 promoter without expression of RASSF10 gene product (*), epidermis (arrow), and connective tissue cells are RASSF10 positive. (k) Isotype control. Bar=50μm (a–c, g–k); 100μm (d); 25μm (e, f). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

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