A comparative study of protein profiling by proteomic analysis in camptothecin-resistant PC3 and camptothecin-sensitive LNCaP human prostate cancer cells.

INTRODUCTION: Drug resistance is a major obstacle for the therapy of prostate cancer, but its underlying mechanisms are not clarified. To detect some candidate marker proteins which may confer resistance to the anticancer drug camptothecin (CPT; DNA topoisomerase 1 inhibitor), the current study deals with the comparative proteomic profiling of CPT-resistant PC3 and CPT-sensitive LNCaP human prostate cancer cell lines which have been widely employed as a useful model to investigate prostate cancer cells. MATERIALS AND METHODS: The global profiling of the protein expression was investigated in CPT-resistant PC3 and CPT-sensitive LNCaP prostate cancer cells using 2-dimensional polyacrylamide gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: 144 proteins were identified and their expression levels were compared between the two cell lines. Four proteins - annexin A1, glutathione-S-transferase (GST) pi, galectin (Gal) 3 and glucose-regulated protein 78/Bip - that are suggested to contribute to the development of drug resistance were found to be preferentially or highly expressed in PC3 cells, whereas LNCaP cells did not show detectable expression of annexin A1, GST-pi and Gal-3. CONCLUSION: The expression level of these proteins and/or mRNAs could be a useful parameter to evaluate the chemotherapy resistance in clinical specimens of prostate cancer.

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation.

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P(1) and S1P(3), as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P(1)/S1P(3) receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT.

Mutations in the CHST6 gene in patients with macular corneal dystrophy: immunohistochemical evidence of heterogeneity.

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive disorder leading to severe visual impairment. The carbohydrate sulfotransferase 6 (CHST6) gene, which encodes the corneal N-acetylglucosamine 6-O-sulfotransferase on 16q22 has been identified as a causative gene for MCD. The purpose of this study was to identify mutations in CHST6 in Japanese patients with MCD and evaluate them by means of immunohistochemistry. METHODS: CHST6 was screened in 7 patients and 45 healthy control subjects. Genomic DNA was isolated, and the open reading frame (ORF) of CHST6 was amplified by polymerase chain reaction (PCR). PCR products were analyzed by direct sequencing and restriction enzyme digestion. Immunohistochemistry with a monoclonal anti-keratan sulfate (KS) antibody was performed on corneas of four patients from three families. RESULTS: Three novel mutations (P204Q, R205L, and R177H) and two previously reported mutations (R211W and A217T) were identified in the ORF of CHST6. P204Q, R205L, and R211W were found to be homozygous and R177H and A217T compound heterozygous with R211W on another allele. Immunohistochemistry revealed that R205L homozygous cornea had negative reactivity against the anti-KS antibody, representing type I MCD, and that R211W homozygous and R211W/A217T compound heterozygous corneas had negative or very weak reactivity in the stroma with antibody positive deposits, which were distinct from any previously reported types. CONCLUSIONS: Two mutations (homozygoous R211W and compound heterozygous R211W/A217T) should be subclassified immunohistochemically into new phenotypes of MCD. This heterogeneity could provide further insights into the pathogenesis of MCD.

Identification of novel mutations of the CHST6 gene in Vietnamese families affected with macular corneal dystrophy in two generations.

PURPOSE: To report the clinical and genetic findings of Vietnamese families affected with macular corneal dystrophy (MCD) in 2 generations. METHODS: Two families, including 7 patients and 3 unaffected members, were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals were used as controls. Genomic DNA was extracted from leukocytes. Analysis of the carbohydrate sulfotransferase (CHST6) gene was performed using polymerase chain reaction and direct sequencing. RESULTS: The typical form of MCD was recognized in family B, in which sequencing of CHST6 gene revealed an nt 1067-1068ins(GGCCGTG) mutation (frameshift after 125V) homozygously in MCD patients and heterozygously in the unaffected members. Family N also showed clinical features of MCD, moderate in the mother but severe in the affected son. Sequencing revealed a single heterozygous Arg211Gln in the mother, compound heterozygous Arg211Gln+ Gln82Stop in the affected son, and heterozygous Arg211Gln mutation in the unaffected members. The identified mutations in these pedigrees were excluded from normal controls. CONCLUSIONS: The novel frameshift and compound heterozygous mutations might be responsible for MCD in the families studied. The phenotypic variation between affected parents and offspring was unclear. In family N, severe MCD phenotype seen in the affected son may be due the fact that he had an early stop codon mutation (Gln82Stop).