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Transcription analysis in the MeLiM swine model identifies RACK1 as a potential marker of malignancy for human melanocytic proliferation.

Egidy G, Julé S, Bossé P, Bernex F, Geffrotin C, Vincent-Naulleau S, Horak V, Sastre-Garau X, Panthier JJ - Mol. Cancer (2008)

Bottom Line: In pig and human samples, the results were similar.In pig metastases, additional nuclear RACK1 did not associate to BDNF expression.In human nevi, the RACK1 signal was low.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UMR955 Génétique Moléculaire et Cellulaire; Laboratoire conventionné CEA n degree 17; Ecole Nationale Vétérinaire d'Alfort, 7 avenue du Général de Gaulle, Maisons-Alfort, F-94704 France. gegidy@pasteur.fr

ABSTRACT

Background: Metastatic melanoma is a severe disease. Few experimental animal models of metastatic melanoma exist. MeLiM minipigs exhibit spontaneous melanoma. Cutaneous and metastatic lesions are histologically similar to human's. However, most of them eventually spontaneously regress. Our purpose was to investigate whether the MeLiM model could reveal markers of malignancy in human melanocytic proliferations.

Results: We compared the serial analysis of gene expression (SAGE) between normal pig skin melanocytes and melanoma cells from an early pulmonary metastasis of MeLiM minipigs. Tag identification revealed 55 regulated genes, including GNB2L1 which was found upregulated in the melanoma library. In situ hybridisation confirmed GNB2L1 overexpression in MeLiM melanocytic lesions. GNB2L1 encodes the adaptor protein RACK1, recently shown to influence melanoma cell lines tumorigenicity. We studied the expression of RACK1 by immunofluorescence and confocal microscopy in tissues specimens of normal skin, in cutaneous and metastatic melanoma developped in MeLiM minipigs and in human patients. In pig and human samples, the results were similar. RACK1 protein was not detected in normal epidermal melanocytes. By contrast, RACK1 signal was highly increased in the cytoplasm of all melanocytic cells of superficial spreading melanoma, recurrent dermal lesions and metastatic melanoma. RACK1 partially colocalised with activated PKCalphabeta. In pig metastases, additional nuclear RACK1 did not associate to BDNF expression. In human nevi, the RACK1 signal was low.

Conclusion: RACK1 overexpression detected in situ in human melanoma specimens characterized cutaneous and metastatic melanoma raising the possibility that RACK1 can be a potential marker of malignancy in human melanoma. The MeLiM strain provides a relevant model for exploring mechanisms of melanocytic malignant transformation in humans. This study may contribute to a better understanding of melanoma pathophysiology and to progress in diagnosis.

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RACK1 expression in normal pig epidermis. (A, C, D) Confocal microscopy analysis of RACK1 protein (green fluorescence), and double labelling for either MITF (A, C) or DCT (D) (red fluorescence) on pig skin. Normal epidermis were from control Meishan minipig (A, B), and MeLiM (C, D). (B) Transmission photograph corresponding to (A). (C) Three dimensional 'orthogonal' slice projection analysis is included: the large central panel shows a single optical slice through which an x axis (green line) and a y axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice (top) and the y, z slice (right) are shown. The blue line represents the position of the central panel image in the z stack. Nuclear counterstaining is shown in blue. Note the RACK1 cytosolic spotty signal on keratinocytes and its absence in the melanocyte indicated by the white dashed line. Dotted lines indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.
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Figure 3: RACK1 expression in normal pig epidermis. (A, C, D) Confocal microscopy analysis of RACK1 protein (green fluorescence), and double labelling for either MITF (A, C) or DCT (D) (red fluorescence) on pig skin. Normal epidermis were from control Meishan minipig (A, B), and MeLiM (C, D). (B) Transmission photograph corresponding to (A). (C) Three dimensional 'orthogonal' slice projection analysis is included: the large central panel shows a single optical slice through which an x axis (green line) and a y axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice (top) and the y, z slice (right) are shown. The blue line represents the position of the central panel image in the z stack. Nuclear counterstaining is shown in blue. Note the RACK1 cytosolic spotty signal on keratinocytes and its absence in the melanocyte indicated by the white dashed line. Dotted lines indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.

Mentions: To explore whether overexpressed RACK1 mRNA was translated, we analysed the cellular distribution of RACK1 protein by confocal microscopy, with double immunostaining for MITF. In control Meishan and healthy MeLiM skins, RACK1 protein was expressed in the epidermis, and found in the cytoplasm of keratinocyte (Figure 3A–D). In MITF-positive (MITF+) melanocytes, RACK1 expression was not detected (Figure 3A, C). Consistent with this, when testing dopachrome tautomerase (DCT), a melanogenic enzyme restricted to melanosomes, double labelling of RACK1 and DCT did not overlap in normal skin (Figure 3D).


Transcription analysis in the MeLiM swine model identifies RACK1 as a potential marker of malignancy for human melanocytic proliferation.

Egidy G, Julé S, Bossé P, Bernex F, Geffrotin C, Vincent-Naulleau S, Horak V, Sastre-Garau X, Panthier JJ - Mol. Cancer (2008)

RACK1 expression in normal pig epidermis. (A, C, D) Confocal microscopy analysis of RACK1 protein (green fluorescence), and double labelling for either MITF (A, C) or DCT (D) (red fluorescence) on pig skin. Normal epidermis were from control Meishan minipig (A, B), and MeLiM (C, D). (B) Transmission photograph corresponding to (A). (C) Three dimensional 'orthogonal' slice projection analysis is included: the large central panel shows a single optical slice through which an x axis (green line) and a y axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice (top) and the y, z slice (right) are shown. The blue line represents the position of the central panel image in the z stack. Nuclear counterstaining is shown in blue. Note the RACK1 cytosolic spotty signal on keratinocytes and its absence in the melanocyte indicated by the white dashed line. Dotted lines indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2387171&req=5

Figure 3: RACK1 expression in normal pig epidermis. (A, C, D) Confocal microscopy analysis of RACK1 protein (green fluorescence), and double labelling for either MITF (A, C) or DCT (D) (red fluorescence) on pig skin. Normal epidermis were from control Meishan minipig (A, B), and MeLiM (C, D). (B) Transmission photograph corresponding to (A). (C) Three dimensional 'orthogonal' slice projection analysis is included: the large central panel shows a single optical slice through which an x axis (green line) and a y axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice (top) and the y, z slice (right) are shown. The blue line represents the position of the central panel image in the z stack. Nuclear counterstaining is shown in blue. Note the RACK1 cytosolic spotty signal on keratinocytes and its absence in the melanocyte indicated by the white dashed line. Dotted lines indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.
Mentions: To explore whether overexpressed RACK1 mRNA was translated, we analysed the cellular distribution of RACK1 protein by confocal microscopy, with double immunostaining for MITF. In control Meishan and healthy MeLiM skins, RACK1 protein was expressed in the epidermis, and found in the cytoplasm of keratinocyte (Figure 3A–D). In MITF-positive (MITF+) melanocytes, RACK1 expression was not detected (Figure 3A, C). Consistent with this, when testing dopachrome tautomerase (DCT), a melanogenic enzyme restricted to melanosomes, double labelling of RACK1 and DCT did not overlap in normal skin (Figure 3D).

Bottom Line: In pig and human samples, the results were similar.In pig metastases, additional nuclear RACK1 did not associate to BDNF expression.In human nevi, the RACK1 signal was low.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UMR955 Génétique Moléculaire et Cellulaire; Laboratoire conventionné CEA n degree 17; Ecole Nationale Vétérinaire d'Alfort, 7 avenue du Général de Gaulle, Maisons-Alfort, F-94704 France. gegidy@pasteur.fr

ABSTRACT

Background: Metastatic melanoma is a severe disease. Few experimental animal models of metastatic melanoma exist. MeLiM minipigs exhibit spontaneous melanoma. Cutaneous and metastatic lesions are histologically similar to human's. However, most of them eventually spontaneously regress. Our purpose was to investigate whether the MeLiM model could reveal markers of malignancy in human melanocytic proliferations.

Results: We compared the serial analysis of gene expression (SAGE) between normal pig skin melanocytes and melanoma cells from an early pulmonary metastasis of MeLiM minipigs. Tag identification revealed 55 regulated genes, including GNB2L1 which was found upregulated in the melanoma library. In situ hybridisation confirmed GNB2L1 overexpression in MeLiM melanocytic lesions. GNB2L1 encodes the adaptor protein RACK1, recently shown to influence melanoma cell lines tumorigenicity. We studied the expression of RACK1 by immunofluorescence and confocal microscopy in tissues specimens of normal skin, in cutaneous and metastatic melanoma developped in MeLiM minipigs and in human patients. In pig and human samples, the results were similar. RACK1 protein was not detected in normal epidermal melanocytes. By contrast, RACK1 signal was highly increased in the cytoplasm of all melanocytic cells of superficial spreading melanoma, recurrent dermal lesions and metastatic melanoma. RACK1 partially colocalised with activated PKCalphabeta. In pig metastases, additional nuclear RACK1 did not associate to BDNF expression. In human nevi, the RACK1 signal was low.

Conclusion: RACK1 overexpression detected in situ in human melanoma specimens characterized cutaneous and metastatic melanoma raising the possibility that RACK1 can be a potential marker of malignancy in human melanoma. The MeLiM strain provides a relevant model for exploring mechanisms of melanocytic malignant transformation in humans. This study may contribute to a better understanding of melanoma pathophysiology and to progress in diagnosis.

Show MeSH
Related in: MedlinePlus