Potential genetic basis of B cell hyperactivation in murine lupus models.

BACKGROUND: Lupus B cells not only produce autoantibodies against nuclear antigens but also provide co-stimulation to T cells. However, there is still a lack of comprehensive understanding of the mechanism underlying lupus B cell hyperactivation. METHODS: This study focuses on the detection of B cell activation status, analysis of early BCR signaling response, DNA sequencing, and quantity determination of BCR signaling regulators in murine lupus models. RESULTS: Our result showed that there is a B cell hyperactivation with a significant elevation of B cell activation markers, and a BCR signaling hyperactivity with an abnormal increase of phosphorylated BCR signaling molecules and cytoplasmic calcium in the early response to BCR crosslinking in B6.Sle1/2/3 lupus mouse. Whole exome sequencing identified a multiple point mutation in the exon of many BCR signaling regulators in common murine lupus models, MRL/lpr, NZM2410, BXSB, NZB, and NZW strains. cNDA sequencing confirmed FcγR2b, Ly9, Pirb, Siglecg, and CD22 BCR signaling regulator variants in B6.Sle1/2/3 lupus mouse, but surface protein expression of these regulators on B cells showed an abnormal increase. CONCLUSION: Our findings support that these BCR signaling regulator variants are potential causative genes of B cell hyperactivation in murine lupus models through their possible functional reduction.

Association of BCSC-1 with human esophageal squamous cell carcinoma.

Breast cancer suppressor candidate-1 (BCSC-1) is a newly identified candidate tumor suppressor gene. BCSC-1 shows decreased levels in a variety of cancer types. In this study, we investigated the association between BCSC-1 and human esophageal squamous cell carcinoma (ESCC). BCSC-1 expression was detected in ESCC and normal tissues adjacent to tumor tissues by Western blot analysis and real-time PCR  as well as immunohistochemistry of paraffin sections. The relationships between BCSC-1 expression and various clinicopathological characteristics were analyzed. Western blot analysis and real-time PCR showed that levels of BCSC-1 protein and mRNA expression in ESCC significantly decreased compared with those in adjacent normal tissues.  Immunohistochemistry exhibited marked reduction of BCSC-1 in 38 of 105 ESCC specimens. Moreover, downregulation of BCSC-1 was associated with the grade of tumor cellular differentiation (P<0.05). These findings indicate that BCSC-1 downregulation in ESCC is associated with carcinogenesis and may play important roles during the process of ESCC cancer development.

Downregulation of PLK1 by RNAi attenuates the tumorigenicity of esophageal squamous cell carcinoma cells via promoting apoptosis and inhibiting angiogenesis.

Polo-like kinase 1(PLK1) is essential for the maintenance of genomic stability during mitosis. PLK1 has been reported to be upregulated in several solid tumors, including esophageal squamous cell carcinoma (ESCC). However, the role of PLK1 in tumorigenesis of ESCC remains undetermined. We used siRNA and lentivirus-mediated PLK1 RNA interference to investigate the tumor suppressor function of PLK1 reduction in ESCC cells. Flow cytometry and Terminal deoxynuleotidyl transferase-mediated nick-end labeling assay in vitro, as well as immunohistochemitry analysis of Caspase-3 and CD31 in s.c. tumor tissue section, were performed. Knock down of PLK1 expression significantly suppressed the ability of ESCC cells to form colonies in plastic and soft agar. PLK1 reduction mediated by lentivirus caused growth suppression of ESCC in nude mice. Caspase-3 upregulation further indicated that dysregulated apoptosis might contribute to reduced tumorigenecity. In particular, downregulation of CD31 suggested that PLK1 reduction-induced angiogenesis inhibition may also contribute, at least in part, to attenuated tumorigenecity. These findings indicate that PLK1 might play roles in tumorigenesis of ESCC and that PLK1 might be a potential gene therapy target in ESCC. Apoptosis induction together with decreased angiogenesis might be involved in the mechanism of tumor suppressor function of RNA interference targeting PLK1.

Tumor suppressor function of BCSC-1 in nasopharyngeal carcinoma.

BCSC-1 is dramatically upregulated in CNE-2L2 human nasopharyngeal carcinoma cells with reduced malignancy (AS cells) and is proposed to be a candidate tumor suppressor gene. We therefore examined the effect of BCSC-1 expression on malignant behaviors of CNE-2L2 cells. Growth in vitro and tumorigenesis in nude mice of wild-type CNE-2L2 cells (W cells) were inhibited by ectopic BCSC-1, and those of AS cells were promoted by BCSC-1 suppression. The tumor suppressor function of BCSC-1 was further confirmed by a study showing that intratumor BCSC-1 injection caused growth suppression of the tumor from W cells inoculated in nude mice. Immunohistochemistry exhibited marked reduction of BCSC-1 expression in 11 of 39 human nasopharyngeal carcinoma specimens. Because BCSC-1 expression was as rich as that in normal cells in the rest of the carcinoma specimens and was poor in CNE-2L2 cells, HNE-1 human nasopharyngeal carcinoma cells with rich BCSC-1 expression were used as a control in the study. No effect of BCSC-1 transfection on growth of the cells was observed. The data suggest that BCSC-1 suppression might play roles in tumorigenesis of some nasopharyngeal carcinomas and that BCSC-1 might be a potential gene therapy target in nasopharyngeal carcinomas with poor BCSC-1 expression. Enhanced aggregation of cells together with increased E-cadherin and alpha-catenin expression and reduced Wnt signaling might be involved in the mechanisms of tumor suppressor function of BCSC-1.

Inhibition of malignant activities of nasopharyngeal carcinoma cells with high expression of CD44 by siRNA.

Since CD44 was found to be significantly inhibited in the human nasopharyngeal carcinoma cell line CNE-2L2 with profound reduction of malignant activities caused by inhibition of alpha-mannosidase Man2c1 gene expression and CD44 has been observed to be involved in many tumor-supporting functions, we studied the association of CD44 expression with the malignant activities of CNE-2L2 cells. Suppression of CD44 gene expression by RNA silencing technique resulted in profound reduction of malignant potential of the cells, including growth in vitro, colony formation, tumorigenesis and metastasis of tumors in nude mice. Direct injection of the adenoviruses harboring and producing siRNA to CD44 into the tumor inoculated with CNE-2L2 cells in nude mice caused inhibition of tumor growth. The data indicate a positive association of CD44 expression with the malignant activities of CNE-2L2 cells and suggest a possible therapeutic effect of direct introduction of siRNA to CD44 into tumors in some human solid tumors with high expression of CD44 gene.

The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin.

Camptothecin is an antitumor agent that kills cells by converting DNA topoisomerase I into a DNA-damaging poison. Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood. We report here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair camptothecin-mediated damage to DNA. The hypersensitivity to camptothecin in the trf4 mutant does not result from elevated expression of DNA topoisomerase I. We show that Trf4 is a nuclear protein whose expression is cell cycle-regulated at a post-transcriptional level. Suppression of camptothecin hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may influence higher order chromosome structure, ZDS1 and ZDS2. We have isolated and sequenced two human TRF4 family members, hTRF4-1 and hTRF4-2. The hTRF4-1 gene maps to chromosome 5p15, a region of frequent copy number alteration in several tumor types. The evolutionary conservation of TRF4 suggests that it may also influence mammalian cell sensitivity to camptothecin.

Analysis of sisomicin binding sites in Micromonospora inyoensis cell wall.

Sisomicin binding sites are located in the cell wall. They are the carboxyl groups of peptidoglycans, which are major components of the cell wall. The carboxyl groups have negative charges which can bind the positively charged amino groups of sisomicin. When the negative charges of the carboxyl groups of the peptidoglycans are changed to neutral or positive charges, sisomicin does not bind to the cell wall. Magnesium ions bind to the cell wall in competition with sisomicin, and have a weak affinity for the cell wall binding sites compared with sisomicin. The binding molar ratio of sisomicin to magnesium ions was approximately 1 to 10.

Blastocoele formation and cell allocation to the inner cell mass and trophectoderm in haploid and diploid pig parthenotes developing in vitro.

The objective of this study was to determine developmental pattern and cell allocation to the inner cell mass and trophectoderm in haploid and diploid embryos following parthenogenetic activation. In vitro matured porcine oocytes were activated by ethanol treatment and cultured in the presence or absence of cytochalasin B for 5 h. The oocytes were then cultured in the NCSU23 for 9 days. The combined treatment with cytochalasin B following ethanol treatment did not increase (p > 0.1) the incidence of activation. The incidence of development to the blastocyst stage was higher (p < 0.05) in the combined treatments of ethanol and cytochalasin B as compared with ethanol treatment alone. The percentage of oocytes with two female pronuclei was higher (p < 0.01) in oocytes treated with cytochalasin B than that in ethanol treatment alone. Treatment with both ethanol and cytochalasin B increased (p < 0.01) the incidence of diploid chromosome spread over just the ethanol treatment alone. The average numbers of total cells and inner cell mass were significantly reduced (p < 0.05) in the ethanol treatment alone as compared with the combined cytochalasin B and ethanol treatment. These results suggested that the ploidy may affect blastocoele formation and cell allocation to inner cell mass and trophectoderm in the pig.

Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.