Caracterização de um novo antígeno tumoral: CTSP-1 / Characterization of a new tumor antigen: CTSP-1

RESUMO

A necessidade de identificar novos antígenos tumorais que possam ser utilizados no tratamento e diagnóstico do câncer, tem levado ao desenvolvimento de técnicas cada vez mais eficientes na detecção dos mesmos. Antígenos de diferentes categorias foram identificados e caracterizados, sendo os antígenos cancer-testis (CT) e os antígenos de diferenciação (CD) os de maior importância clínica, dado seu restrito padrão de expressão. Utilizando uma estratégia de alinhamento de seqüências expressas no genoma humano, identificamos um novo transcrito localizado no cromossomo 21, denominado CTSP-1, que apresenta alta similaridade com o antígeno tumoral NY-BR-1... Além disso, avaliamos seu padrão de expressão em diferentes tecidos normais, linhagens celulares tumorais e amostras de tumores de pacientes... Através de immunoblotting foi possível a identificação de uma banda específica de 22kDa, correspondente ao peso esperado da proteína CTSP-1 em extrato total de testículo normal. Em seguida, através da imunohistoquímica, verificamos uma marcação preferencial nas espermatogônias e células de Leydig de testículo normal. Nos tecidos com amostras pareadas normal/tumor (mama e próstata), apenas as amostras tumorais foram fortemente marcadas. Posteriormente, a proteína CTSP-1 recombinante foi utilizada na investigação de anticorpos específicos em plasma de pacientes com câncer. Aproximadamente 150 amostras foram analisadas, das quais 20% apresentaram resposta imune humoral contra a proteína CTSP-1. Estes resultados revelam que o CTSP-1 é um novo antígeno tumoral da categoria dos CTs, com expressão restrita a tumor e testículo e com alta imunogenicidade em pacientes com câncer...(AU)

ABSTRACT

The need to identify new tumor antigens to be used in cancer treatment and diagnosis has lead to the development of efficient techniques for this purpose. Antigens from different categories have been identified and characterized and, among those, the cancer-testis (CT) and the cancer differentiation (CD) antigens are of the greatest clinicai interest dueto their restricted expression partem. Using alignments between expressed sequences and the Human Genome Sequence, we identified a new gene located on chromosome 21, named CTSP-1. This gene has a high similarity to the tumor antigen NY-BR-1, which encodes for a tissue specific transcription factor and is a potential target for cancer immunotherapy. In order to verify ifthe CTSP-1 gene is really a new tumor antigen, we performed its complete characterization. Using different techniques, we were able to obtain the complete sequence of the CTSP-1 gene and to identify different alternative polyadenilation and splicing forms. Moreover, we analyzed the CTSP-1 expression pattern in normal tissues, tumor cell lines and tumor samples. CTSP-1 showed a restricted expression pattern, being expressed only in testis among normal tissues, in different tumor celllines (9/22) and in different tumor types (7 4/178), which matches with the expression pattern of Cancer-Testis antigens. Afterwards, the recombinant CTSP-1 protein was expressed in a heterologous system and used for the generation of polyclonal antibody in mice. This antiboby was used in immunoblotting and immunohistochemistry experiments for the detection of CTSP-1 protein in normal testis and paired normal and tumor samples from breast and prostate. Using immunoblotting, a 22kDa specific band was identified in testis total protein extract, corresponding to the expected molecular weight of the CTSP-1 protein. Using immunohistochemistry, we verified the preferential staining of germ cells and Leydig cells in normal testis. Among tissues with paired normal/tumor samples, only tumor samples were strongly stained. The CTSP-1 recombinant protein was also used in the search for specific antibodies in plasma from cancer patients. Approximately 150 samples were analyzed, of which 20% showed a humoral immune response against the CTSP-1 protein. Taken together, these results confirm that the CTSP-1 gene is a new tumor antigen from the Cancer-Testis category, with restricted expression in testis and tumors and with high immunogenicity in cancer patients (AU)