RESUMO
Cancer/testis antigens (CTA) are a heterogeneous group of antigens that are expressed preferentially in tumor cells and testis. Based on this definition the human membrane-associated phospholipase A1 beta (lipase family member I, LIPI) has been identified as CTA. The high homology of LIPI and the membrane-associated phospholipase A1 alpha (lipase family member H, LIPH) suggests that both genes are derived from a common ancestor by gene duplication. In contrast to human LIPI, human LIPH is expressed in several tissues. LIPI sequences have only been identified in mammals. Here, we describe the identification of LIPI in non-mammalian vertebrates. Based on the conserved genomic organization of LIPI and LIPH we identified sequences for both lipases in birds and fishes. In all vertebrates the LIPI locus is neighbored by a member of the RNA binding motif (RBM) family, RBM11. By sequencing of reverse transcriptase-polymerase chain reaction products we determined the sequences of LIPI and LIPH messenger RNA from broilers. We found that the sequence homology between LIPI and LIPH is much higher in non-mammalian species than in mammals. In addition, we found broad expression of LIPI in broilers, resembling the expression profile of LIPH. Our data suggest that LIPI is a CTA only in mammalian species and that the unique sequence features of the mammalian LIPI/RBM11 locus have evolved together with the CTA-like expression pattern of LIPI.
Assuntos
Antígenos de Neoplasias/genética , Galinhas/genética , Isoenzimas/genética , Fosfolipases A1/genética , Animais , Sequência de Bases , Primers do DNA/genética , Evolução Molecular , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da EspécieRESUMO
The prognosis for patients with advanced stages of Ewing family tumors (EFT) is very poor. EFT express high levels of phosphatidic acid specific membrane-associated phospholipase A1 beta (lipase I, LIPI). LIPI is a cancer/testis antigen and the high tumor specificity suggests that LIPI might be an attractive target for new diagnostic and/or therapeutic developments. By using reverse transcriptase-polymerase chain reaction (RT-PCR), we observed simultaneous presence of multiple LIPI transcript variants in EFT. We cloned and sequenced these transcript variants from EFT cell lines. Sequence analysis indicated that all transcript variants were derived by alternative splicing. Homology modeling of corresponding protein structures suggested that different transcript variants differ in their regulatory lid domains. In addition, expression of receptors for lysophosphatidic acid (LPA) was analyzed in a panel of EFT cell lines by RT-PCR. We observed that EFT cell lines expressed high levels of LPA receptors. Different LIPI transcript variants present in EFT might be involved in the pathogenesis of EFT by signaling via these LPA receptors.
Assuntos
Processamento Alternativo/genética , Antígenos de Neoplasias/genética , Membrana Celular/enzimologia , Regulação Neoplásica da Expressão Gênica , Fosfolipases A1/genética , Receptores de Ácidos Lisofosfatídicos/genética , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/genética , Linhagem Celular Tumoral , Éxons/genética , Humanos , Lipase/genética , Lipase/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Homologia Estrutural de ProteínaRESUMO
Despite improvements in the treatment of patients with Ewing family tumors (EFT), the prognosis for patients with advanced disease is still unsatisfactory. Recently, we identified lipase I as an EFT-associated gene that might be interesting for the development of new immunological or pharmacological treatment strategies. Lipase I is a member of the large protein superfamilies of alpha/beta hydrolases and serine hydrolases. In the present paper we describe high expression of another member of these superfamilies in EFT. By DNA microarray data base mining we found exceptional high expression of alpha/beta hydrolase domain containing 6 (ABHD6) in EFT but not in other sarcomas. Expression of ABHD6 in EFT correlated with expression of another EFT-associated gene, aristaless. Analysis of ABHD6-associated GGAA microsatellites revealed shorter microsatellites in EFT with lack of ABHD6 expression. ABHD6 homologues were found in varying chordata but not in other animal species. Based on homology modeling we predicted the 3D-structure of ABHD6, which shows high similarity with bacterial homoserine transacetylases. High expression of ABHD6 in EFT in comparison to normal tissues and other tumors suggests that ABHD6 might be an interesting new diagnostic or therapeutic target for EFT. However, knock down of ABHD6 in EFT cells did not inhibit tumor cell growth.
Assuntos
Monoacilglicerol Lipases/fisiologia , Sarcoma de Ewing/enzimologia , Neoplasias Ósseas , Hidrolases de Éster Carboxílico/genética , Linhagem Celular Tumoral , Evolução Molecular , Humanos , Lipase/genética , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/genética , Prognóstico , Sarcoma de Ewing/mortalidadeRESUMO
BACKGROUND: Ewing family tumours (EFT) are the second most common bone tumours in children and adolescents. In the majority of EFT, EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukaemia virus integration 1) fusion proteins can be detected and EWSR1-FLI1 substantially contributes to the malignant phenotype of EFT. Therefore, inactivation of EWSR1-FLI1 is an interesting strategy for EFT therapy. MATERIALS AND METHODS: A ribozyme with specificity for EWSR1-FLI1 was developed and the activity in vitro was investigated. Synthetic RNAs corresponding to EWSR1-FLI1 were used as substrates. In addition, the total RNA from EFT cells was used as substrate and the rapid amplification of cDNA ends method for the detection of the cleavage products was used. RESULTS: The ribozyme cleaved the synthetic RNA in a sequence specific manner with high efficiency in vitro. Furthermore, the expected cleavage products were detected after digestion of the total cellular RNA with this ribozyme. A point mutation in the catalytic centre of the ribozyme abolished enzymatic activity. CONCLUSION: The RNA corresponding to EWSR1-FLI1 is accessible for ribozyme mediated inactivation and ribozymes are able to cleave EWSR1-FLI1 specific RNA in the presence of a high background of normal cellular RNAs.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Ligação a Calmodulina/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Catalítico/farmacologia , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Sequência de Bases , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ligação a Calmodulina/metabolismo , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , RNA Catalítico/síntese química , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
BACKGROUND: Cancer/testis antigens (CTA) represent a heterogeneous group of antigens expressed nearly exclusively in tumour cells and testis. Recently, we identified phospholipase A1 beta (a CTA also known as lipase member I, LIPI) as a gene with high expression in Ewing family tumours (EFT). In the present paper we analyzed expression of LIPI in a panel of normal tissues and tumour samples. PROCEDURE: The expression of CTA in EFT and normal tissues was analyzed by using DNA microarray datasets. Expression of LIPI in EFT, a panel of other tumour samples, and normal tissues was analyzed by using RT-PCR and quantitative RT-PCR. RESULTS: LIPI was expressed in EFT samples but not in other investigated tumour samples. Expression of LIPI in normal tissues was restricted to testis and thyroid. However, expression in these tissues was low compared with EFT. Interestingly testis as well as thyroid expressed all analyzed EFT-associated transcripts, suggesting that these tissues harbour a small cell population with molecular features of EFT. The sensitivity of the LIPI RT-PCR was similar to the sensitivity of the conventional EWSR1-FLI1 RT-PCR, suggesting that LIPI might be useful as additional diagnostic target structure. CONCLUSIONS: The human cancer/testis antigen LIPI is highly expressed in Ewing family tumours and can be easily detected by RT-PCR or quantitative RT-PCR. LIPI might be an interesting target for the development of future diagnostic tools or treatment strategies.
