ABSTRACT
BACKGROUND: Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). RESULTS: Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. CONCLUSION: The majority of identified differentially expressed genes in cutaneous SCC were previously not described.
Subject(s)
Carcinoma, Squamous Cell/genetics , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Skin Neoplasms/genetics , Adolescent , Aged , Female , Humans , Male , Middle Aged , Multigene Family/genetics , RNA, Messenger/geneticsABSTRACT
Carcinogenesis is a multi-step process resulting from the accumulation of genetic mutations and subsequently leading to dysregulated genes, but the number and identity of differentially expressed genes in cutaneous squamous cell carcinoma (SCC) is unknown at present. In order to identify dysregulated genes, we examined the relative mRNA expression present in cutaneous SCC and its precursor lesion actinic keratosis (AK) by comparison to normal skin. Snap frozen biopsies from 20 specimens of normal skin, 10 AK, and 10 cutaneous SCC were examined. Total-RNA was extracted, reversely transcribed, and 14 genes were investigated using gene-specific intron-flanking primers and quantitative real-time reverse transcription PCR. Specificity was confirmed by sequencing of the PCR amplicons. Ten of 14 genes were significantly dysregulated in AK and/or cutaneous SCC by comparison to normal skin. The genes CNN2, COX4I1, COX5B, COX7C, CRLF3, CTSC, NDRG1, and LMNA showed increased expression in skin cancer (p < 0.02), while RPL15 and LGTN were down-regulated (p < 0.03). The genes differentially expressed during skin carcinogenesis may prove useful in order to understand the origin and progression of cutaneous SCC and for diagnostic approaches.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Keratosis/genetics , Neoplasm Proteins/genetics , Skin Neoplasms/genetics , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/pathology , Disease Progression , Female , Gene Expression Profiling , Humans , Keratosis/pathology , Male , Middle Aged , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/pathologyABSTRACT
Eukaryotic translation initiation factor (eIF-5A) is a highly conserved and essential protein that contains the unique amino acid hypusine. The first step in the post-translational biosynthesis of hypusine, the transfer of an aminobutyl moiety from the polyamine substrate spermidine to the -amino group of a specific lysine residue in the eIF-5A precursor, is catalyzed by the enzyme deoxyhypusine synthase. A cDNA encoding a protein homologous to eIF-5A was isolated by plaque hybridization from a cDNA library of Plasmodium falciparum. The cloned cDNA contains an open reading frame encoding a protein of 161 amino acids, which shares a high sequence identity with other eukaryotic eIF-5A sequences. A phylogenetic tree constructed with eIF-5A from P. falciparum and 16 other eIF-5A sequences of eukaryotic and archaeal origin reveals that plasmodial eIF-5A together with other apicomplexan eIF-5A show a higher degree of homology to plant proteins than to animal and fungal sequences. The plasmodial eIF-5A gene was expressed as a six-histidine tagged fusion protein in Escherichia coli. Radioactive incorporation studies with [1,8-3H] spermidine indicated that this protein can serve as a substrate for human deoxyhypusine synthase. Results of quantitative real-time PCR studies with synchronized erythrocytic stages of P. falciparum revealed no significant induction or downregulation but only some variation in the expression level of plasmodial eIF-5A in ring, trophozoite and schizont stage.
