BACKGROUND: Melanocortin-1 receptor (Mc1r), a key signaling receptor for melanogenesis, has been reported to mediate migration of B16F10 melanoma cells. Interestingly, this activity appears to be a part of the constitutive signaling of Mc1r. METHODS: We carried out small interfering RNA-mediated knock-down of Mc1r on murine melanoma B16F10 cells and performed microarray analysis to characterize changes in the gene expression profile. RESULTS: We isolated 22 and four genes whose expression decreased and increased, respectively, by 2.5-fold or higher as the result of Mc1r knock-down. Several down-regulated genes have been proposed to be involved in cell migration. Among these genes are several members of the chemokine gene family. CONCLUSION: We provide a gene set for further functional analyses of Mc1r. The Mc1r target genes we present may be particularly relevant for understanding the ligand-independent activity of Mc1r. Further examination of the mode of action may lead to novel strategies in regulating the migration and metastasis of melanoma cells.
Cell Movement ; Chemokines ; Gene Expression Regulation* ; Genes, vif ; Humans ; Melanoma ; Microarray Analysis ; Neoplasm Metastasis ; Receptor, Melanocortin, Type 1* ; Transcriptome

BACKGROUND: Vitiligo is a skin disease that is characterized by the loss of cutaneous pigmentation. alpha-Melanocyte stimulating hormone (alpha-MSH) is a neuroimmunomodulating peptide derived from proopiomelanocortin, and melanocortin-1 receptor (MC1R) is a surface receptor which is expressed by several other cutaneous cells including melanocyte and keratinocyte. Both of them have been known to be the main physiologic regulator for integumental pigmentation. OBJECTIVE: To evaluate the expression pattern of alpha-MSH and MC1R in the epidermis of vitiligo patients. METHODS: Specimens were obtained in lesional, perilesional and non-lesional skin in 10 patients with vitiligo and from 3 normal persons by the punch biopsy. And then, indirect immunofluorescence was done to show the pattern of expression of alpha-MSH and MC1R. RESULTS: Pattern of expression between alpha-MSH and MC1R was nearly the same. In vitiligo patients with stable disease state (7 of 10), the expression of alpha-MSH and MC1R in the non-lesional skin was more prominent than that in lesional area. In vitiligo patients with active disease state (3 of 10), the expression of alpha-MSH and MC1R in the lesional skin was more prominent than that in non-lesional area. CONCLUSION: Between the stable and active vitiligo patients, there was a different pattern of expression of alpha-MSH and MC1R in the lesional skin.
alpha-MSH ; Biopsy ; Epidermis* ; Fluorescent Antibody Technique, Indirect ; Humans ; Keratinocytes ; Melanocytes ; Pigmentation ; Pro-Opiomelanocortin ; Receptor, Melanocortin, Type 1* ; Skin ; Skin Diseases ; Vitiligo*

PURPOSE: Gastric cancer is one of the most prevalent cancers worldwide. 5-fluorouracil (5-FU) and cisplatin are the most commonly used drugs for the treatment of gastric cancer. However, a significant number of tumors often fail to respond to chemotherapy. MATERIALS AND METHODS: To better understand the molecular mechanisms underlying drug resistance in gastric cancer the gene expression in gastric cancer cells, which were either sensitive or resistant to 5-FU and cisplatin, were examined using cDNA microarray analysis. To confirm the differential gene expression, as determined using the microarray, semiquantitative RT-PCR was performed on a subset of differentially expressed cDNAs. RESULTS: 69 and 45 genes, which were either up-regulated (9 and 22 genes) or down-regulated (60 and 25 genes), were identified in 5-FU- and cisplatin-resistant cells, respectively. Several genes, such as adaptor-related protein complex 1 and baculoviral IAP repeat-containing 3, were up-regulated in both drug-resistant cell types. Several genes, such as the ras homolog gene family, tropomyosin, tumor rejection antigen, protein disulfide isomerase-related protein, melanocortin 1 receptor, defensin, cyclophilin B, dual specificity phosphatase 8 and hepatocyte nuclear factor 3, were down-regulated in both drug-resistant cell types. CONCLUSION: These findings show that cDNA microarray analysis can be used to obtain gene expression profiles that reflect the effect of anticancer drugs on gastric cancer cells. Such data may lead to the assigning of signature expression profiles of drug-resistant tumors, which may help predict responses to drugs and assist in the design of tailored therapeutic regimens to overcome drug resistance.
Adaptor Protein Complex 1 ; Cisplatin* ; Cyclophilins ; DNA, Complementary* ; Drug Resistance ; Drug Therapy ; Dual-Specificity Phosphatases ; Fluorouracil* ; Gene Expression* ; Hepatocytes ; Humans ; Oligonucleotide Array Sequence Analysis* ; Receptor, Melanocortin, Type 1 ; Stomach Neoplasms* ; Transcriptome ; Tropomyosin

OBJECTIVETo assess the association between single nucleotide polymorphisms (SNPs) of melanocortin-1 receptor gene (MC1R) and freckles in Chinese Han population from Chengdu.

METHODSTwenty randomly selected samples were used to select SNPs of the MC1R gene through DNA sequencing. Pyrosequencing in combination with DNA pooling technique was used to assess allelic frequencies of the selected SNPs in 111 individuals with freckles and 124 normal controls. Representative SNPs were selected based on their functional implications and minimum allele frequency (MAF> 0.05). Genotype of the SNPs were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or pyrosequencing.

RESULTSBased on results of DNA sequencing and pyrosequencing, 4 SNPs (rs2228479, rs885479, rs33932559 and rs2228478) were selected to determine the genotype for each sample. Comparison of genotypic and allelic frequencies of the 4 SNPs with χ (2) test has found no significant difference between the two groups (P> 0.05). For rs33932559, the frequencies of T allele were respectively 90.09% and 91.94% for individuals with freckles and normal controls. For rs2228479 and rs2228478, the frequencies of G and A allele were both about 77%. For rs885479, the frequency of T allele was about 60%. None of the above 3 SNPs showed a significant difference between the two groups in terms of allelic or genotypic frequencies.

CONCLUSIONNo association between the selected SNPs of MC1R gene has been found with development of freckles for the selected Chinese Han population from Chengdu.

Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; China ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Melanosis ; genetics ; Middle Aged ; Polymorphism, Single Nucleotide ; Receptor, Melanocortin, Type 1 ; genetics ; Young Adult

Binding activity and biologic effect of a novel alpha-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3. 1/ myc-his(-) B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection (900 microg x mL(-1)) for subsequent bioassay analysis. K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were obtained in competition with [125I]-NDP-MSH for binding studies. The cyclic AMP level was tested by using [3H]-cyclic AMP kit. It is showed that K(i) values of the novel alpha-MSH analogue for MC1, MC3, MC4, and MC5 receptors were (0.159 +/- 0.040), (35.430 +/- 6.743), (19.293 +/- 2.780) and (2.230 +/- 0.670) nmol L(-1), respectively. Its EC50 values for MC1, MC3, MC4, and MC5 receptors were (0.45 +/- 0.07), (7.80 +/- 0.65), (2.55 +/- 0.23) and (0.33 +/- 0.09) nmol L(-1), respectively. In these tests, the novel alpha-MSH analogue is a MC1R and MC5R selective agonist.
Amino Acid Sequence ; Binding, Competitive ; Cell Line ; Cell Line, Tumor ; Cyclic AMP ; metabolism ; Genetic Vectors ; Humans ; Iodine Radioisotopes ; Kinetics ; Molecular Sequence Data ; Plasmids ; genetics ; Radioligand Assay ; Receptor, Melanocortin, Type 1 ; agonists ; genetics ; metabolism ; Receptors, Corticotropin ; agonists ; genetics ; metabolism ; Receptors, Melanocortin ; agonists ; genetics ; metabolism ; Transfection ; Tritium ; alpha-MSH ; analogs & derivatives ; chemistry ; metabolism ; pharmacology