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Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker.

Salazar-Onfray F, López M, Lundqvist A, Aguirre A, Escobar A, Serrano A, Korenblit C, Petersson M, Chhajlani V, Larsson O, Kiessling R - Br. J. Cancer (2002)

Bottom Line: Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes.Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas.Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro.

View Article: PubMed Central - PubMed

Affiliation: Disciplinary Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Av. Independencia 1027, Santiago, Chile. fsalazar@machi.med.unchile.cl

ABSTRACT
The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT-PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a 'therapeutic window' to be exploited in cancer immunotherapy.

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Expression of MC1R in melanoma cell lines. Upper panel: Cell lines (a) C1R-A2, (b) C1R-A2 neo, (c) C1R-A2/MC1R(1), (d) C1R-A2/MC1R(2), (e) FMS, (f) OCM1, (g) OCM3 were analysed by Western blot for the presence of the 37 kD band corresponding to MC1R. Lower panel: DFB melanoma. (A), FM55 melanoma (B), BL melanoma (C), C1R-A2 LCL line as a negative control (D), and C1R-A2/MC1R (E) were fixed and incubated with mAb MP4-1B7 directly (left) or after permeabilisation with digitonin (right) and then stained with PE-conjugate anti-IgG mAbs and analysed by FACS as described in Materials and Methods. The experiments were performed twice with similar results.
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fig1: Expression of MC1R in melanoma cell lines. Upper panel: Cell lines (a) C1R-A2, (b) C1R-A2 neo, (c) C1R-A2/MC1R(1), (d) C1R-A2/MC1R(2), (e) FMS, (f) OCM1, (g) OCM3 were analysed by Western blot for the presence of the 37 kD band corresponding to MC1R. Lower panel: DFB melanoma. (A), FM55 melanoma (B), BL melanoma (C), C1R-A2 LCL line as a negative control (D), and C1R-A2/MC1R (E) were fixed and incubated with mAb MP4-1B7 directly (left) or after permeabilisation with digitonin (right) and then stained with PE-conjugate anti-IgG mAbs and analysed by FACS as described in Materials and Methods. The experiments were performed twice with similar results.

Mentions: The specificity of the mAbs MP1-1B7 and MP1-1C11, previously described to be specific for the extracellular domain of MC1R (Thörnvall et al, 1997), was confirmed by Western blot analysis on melanoma lines and EBV immortalized B cell lines (LCL). Three melanoma cell lines (FMS, OCM3 and OCM1) were positive for a 37 kD protein, which corresponds to the expected size of MC1R, while the LCL line C1R-A2 was negative. C1R-A2 transiently transfected with the vector pRC/CMV-hMCIR was positive for MC1R, confirming the specificity of the antibody (Figure 1Figure 1


Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker.

Salazar-Onfray F, López M, Lundqvist A, Aguirre A, Escobar A, Serrano A, Korenblit C, Petersson M, Chhajlani V, Larsson O, Kiessling R - Br. J. Cancer (2002)

Expression of MC1R in melanoma cell lines. Upper panel: Cell lines (a) C1R-A2, (b) C1R-A2 neo, (c) C1R-A2/MC1R(1), (d) C1R-A2/MC1R(2), (e) FMS, (f) OCM1, (g) OCM3 were analysed by Western blot for the presence of the 37 kD band corresponding to MC1R. Lower panel: DFB melanoma. (A), FM55 melanoma (B), BL melanoma (C), C1R-A2 LCL line as a negative control (D), and C1R-A2/MC1R (E) were fixed and incubated with mAb MP4-1B7 directly (left) or after permeabilisation with digitonin (right) and then stained with PE-conjugate anti-IgG mAbs and analysed by FACS as described in Materials and Methods. The experiments were performed twice with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2376124&req=5

fig1: Expression of MC1R in melanoma cell lines. Upper panel: Cell lines (a) C1R-A2, (b) C1R-A2 neo, (c) C1R-A2/MC1R(1), (d) C1R-A2/MC1R(2), (e) FMS, (f) OCM1, (g) OCM3 were analysed by Western blot for the presence of the 37 kD band corresponding to MC1R. Lower panel: DFB melanoma. (A), FM55 melanoma (B), BL melanoma (C), C1R-A2 LCL line as a negative control (D), and C1R-A2/MC1R (E) were fixed and incubated with mAb MP4-1B7 directly (left) or after permeabilisation with digitonin (right) and then stained with PE-conjugate anti-IgG mAbs and analysed by FACS as described in Materials and Methods. The experiments were performed twice with similar results.
Mentions: The specificity of the mAbs MP1-1B7 and MP1-1C11, previously described to be specific for the extracellular domain of MC1R (Thörnvall et al, 1997), was confirmed by Western blot analysis on melanoma lines and EBV immortalized B cell lines (LCL). Three melanoma cell lines (FMS, OCM3 and OCM1) were positive for a 37 kD protein, which corresponds to the expected size of MC1R, while the LCL line C1R-A2 was negative. C1R-A2 transiently transfected with the vector pRC/CMV-hMCIR was positive for MC1R, confirming the specificity of the antibody (Figure 1Figure 1

Bottom Line: Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes.Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas.Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro.

View Article: PubMed Central - PubMed

Affiliation: Disciplinary Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Av. Independencia 1027, Santiago, Chile. fsalazar@machi.med.unchile.cl

ABSTRACT
The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT-PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a 'therapeutic window' to be exploited in cancer immunotherapy.

Show MeSH
Related in: MedlinePlus