RESUMO
A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.
Assuntos
L-Serina Desidratase/química , L-Serina Desidratase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , L-Serina Desidratase/genética , Neoplasias Pulmonares/enzimologia , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Multidrug resistance (MDR) is a major impediment to successful chemotherapy for lung cancer. Overexpression of multidrug resistance-associated protein 1 (MRP1) appears to be involved in MDR development in lung cancer cells. A number of chemotherapeutic agents including doxorubicin (DOX) were reported to induce MRP1 expression in human lung cancer cells. In our study, we investigated the mechanism by which DOX induces MRP1 expression in human small cell lung cancer (SCLC) cell lines, GLC4 and NCI-H82. These cells expressed MRP1 protein at low levels and were sensitive to DOX. Doxorubicin at 50 nM induced a marked increase in MRP1 expression in 24 hr, and stimulated c-jun N-terminal kinase (JNK) activity. Treatment with a JNK inhibitor, SP600125, significantly inhibited MRP1 induction. Furthermore, transfection with JNK1 and JNK2 antisense oligonucleotides markedly inhibited DOX-induced MRP1 expression. Chromatin immunoprecipitation assays revealed an enhanced recruitment of phosphorylated c-jun to the MRP1 promoter containing the AP-1 site upon DOX stimulation, which was inhibited by pretreatment with SP600125. Surprisingly, GLC4 cells exposed to DOX for 24 hr maintained increased MRP1 expression and resistance to DOX for at least 3 weeks. Pretreatment with SP600125 before DOX stimulation blocked the appearance of the MDR phenotype as well as MRP1 induction in GLC4 cells. These findings suggest that JNK activation may play an essential role for the induction of MRP1 protein in human SCLC cells by chemotherapeutic agents and that combined treatment of a JNK inhibitor with anticancer drugs may prevent the development of MDR by the abrogation of MRP1 induction.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Carcinoma de Células Pequenas/tratamento farmacológico , Doxorrubicina/uso terapêutico , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Carcinoma de Células Pequenas/metabolismo , Imunoprecipitação da Cromatina , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A 68-year-old man was admitted to our hospital because of muscle weakness. A complete medical examination led to a diagnosis of small cell lung carcinoma (SCLC) with Lambert-Eaton myasthenic syndrome (LEMS) and the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Four courses of chemotherapy (carboplatin + etoposide) and one of radiotherapy with a total dose of 45 Gy to the mediastinum were performed and resulted in a partial response in the SCLC. After the second course of chemotherapy, the serum level of antivoltage-gated Ca2+ channel (VGCC) antibody decreased from 190 pg/ml to 120 pg/ml. Marked improvement of the muscle weakness was recognized only after 3 courses of chemotherapy. The patient, who had had difficulty in standing, recovered enough to be able to climb stairs after 4 courses of chemotherapy. Marked improvement of LEMS was achieved by treatment for small cell lung carcinoma.