Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro.

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 microM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 +/- 3.9% in HNE1-LMP1 cells vs 37.89 +/- 4.9% in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 +/- 3.5 vs 65.87 +/- 5.9%), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 +/- 3.7 and 27.91 +/- 2.1%), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 +/- 3.9 vs 29.19 +/- 6.27%) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.

Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 æM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 ± 3.9 percent in HNE1-LMP1 cells vs 37.89 ± 4.9 percent in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 ± 3.5 vs 65.87 ± 5.9 percent), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 ± 3.7 and 27.91 ± 2.1 percent), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 ± 3.9 vs 29.19 ± 6.27 percent) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.

A strategy for probing the function of noncoding RNAs finds a repressor of NFAT.

Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.

Latent membrane protein-1 of Epstein - Barr virus increases sensitivity to arsenic trioxide-induced apoptosis in nasopharyngeal carcinoma cell.

BACKGROUND AND AIM: Epstein - Barr virus (EBV)-encoded LMP1 is suggested to have an important role in the pathogenesis and development of nasopharyngeal carcinoma (NPC). Our previous study showed that As2O3 exhibited growth inhibition of NPC in animal model. Here, we further explore whether LMP1 is involved in As2O3 anticancer effects in NPC cell line. METHODS: Both the stable expressing LMP1 cell line HNE1-LMP1 and its parental cell line HNE1 without LMP1 expression were used as in vitro models to assess arsenic trioxide effect. Both cell lines were treated with As2O3 for 72 h. The median inhibition concentration (IC50) was assessed by the MTT assay. Apoptosis was observed by phase-contrast microscopy and TUNEL staining. The alteration of telomere lengths was detected by Southern blotting. RESULTS: IC50 for As2O3 in HNE1-LMP1 cells and HNE1 cells was 2.22 and 5.09 micromol/L, respectively. After exposure to 2 and 4 micromol/L As2O3 for 72 h, the apoptotic index in HNE1-LMP1 was 26.27 -/+ 1.3 and 49.13 -/+ 1.4%, respectively. On the contrary, in HNE1 cells the apoptotic index was 12.6 -/+ 0.9 and 33.20 -/+ 1.3%, respectively. As compared with parental cell line HNE1, HNE1-LMP1 cells were more sensitive to growth inhibition and apoptosis (p < 0.001). The elongation of telomere length was also found in HNE1-LMP1 cells. Meanwhile, longer telomeres in HNE1-LMP1 cells failed to maintain telomere stabilization, instead, it prone to be shortened when exposure to As2O3, as comparing with HNE1 cells. CONCLUSION: LMP1 plays important role in enhancing NPC cell response to As2O3. The elongation of telomere length induced by LMP1 may contribute to the mechanisms of As2O3 sensitivity.

[Research progress in gene engineered antibodies].

Gene engineered antibodies(GEAs) have been applied in a lot of aspects with the great development of the technique of GEAs. The technique of GEAs such as ribosome display and phage surface display and their application progress were summarized in this review.

Sequencing of the ribosomal intergenic spacer region for strain identification of Porphyromonas gingivalis.

The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivalis were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalis ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison.

A role for FADD in T cell activation and development.

FADD is a cytoplasmic adapter molecule that links the family of death receptors to the activation of caspases during apoptosis. We have produced transgenic mice expressing a dominantly interfering mutant of FADD, lacking the caspase-dimerizing death effector domain, as well as mice overexpressing the poxvirus serpin, CrmA, an inhibitor of caspases downstream of FADD. While thymocytes from either line of mice were completely protected from CD95-dependent cytotoxicity, neither transgene afforded protection from apoptosis induced during thymocyte selection and neither led to the lymphoproliferative disorders associated with deficiencies in CD95. However, in FADD dominant negative (FADDdd) mice, early thymocyte development was retarded and peripheral lymphocyte pools were devoid of normal populations of T cells. We show that thymocytes and peripheral T cells from FADDdd display signaling anomalies, implying that FADD plays a previously uncharacterized role in T cell development and activation.

Hepatoma-associated nonhistone chromosomal proteins are present in active chromatin.

Recently a group of nonhistone proteins with molecular weights ranging from 180-200 K were discovered which are associated with rat hepatoma chromatin specifically (Burkhardt et al., Biochim. Biophys. Acta 781, 165-172, 1984). These hepatoma-associated nonhistone proteins appeared and increased in rats treated with a hepatocarcinogen. Two approaches were used in this study to investigate whether the hepatoma-associated nonhistone chromosomal proteins are present in actively transcribed regions. We found that the limited DNase I digestion of Morris hepatoma 7777 chromatin released antigenic proteins not detected in normal liver chromatin digests. The association of antigenic nonhistone proteins with nuclear matrices was also studied. Using immunoblot analysis of nuclear matrices and total chromatin, the antigenic nonhistone chromosomal proteins were determined. Hepatoma-associated nonhistone protein antigens were extensively concentrated in the nuclear matrices. In the present study, the transcriptionally-active alpha-fetoprotein gene and the nontranscribed beta-globin gene were used as gene markers to determine the transcriptionally active chromatin region. Data presented in this paper indicate that hepatoma-associated NHPs are localized in active chromatin.

Monoclonal antibodies to nonhistone proteins associated with human colon cancer nuclear matrix.

Hybridoma technology was applied in an effort to create highly specific probes for nonhistone proteins associated with human colon cancer nuclear matrix. Three stable monoclonal antibodies producing cloned cells No. 39, 54 and 58 are described here. All these antibodies showed high reactivity with human colon tumor nuclear matrix. Both antibodies No. 39 and 58 showed an extensive cross reactivity at high concentration of normal colon nuclear matrix. The antigens were determined to be a heterogeneous group of proteins with a major antigen of molecular weight of 140,000 for antibody No. 54 subclone 54-c-5-6 and two major antigens of molecular weight for 105,000 and 116,000 for antibody No. 39 subclone 39-d-11-12. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge method demonstrated their presence in the nuclei.

Colon adenocarcinoma-associated nuclear antigens.

Colon adenocarcinoma-associated chromosomal nonhistone proteins were determined in rat colon adenocarcinoma chromatin and in colon chromatin of Sprague-Dawley rats treated with 1,2-dimethylhydrazine (DMH) by the enzyme-linked immunosorbent assay method. The specific nuclear proteins are tissue-specific oncofetal antigens. They were also detected in fetal rat colon chromatin but not in adult rat colon chromatin. The specific tumor-associated nonhistone proteins were identified by immunoblot. The molecular weight ranges of these proteins were 79,000-89,000 and 40,000-56,000 daltons. Administration of DMH to Sprague-Dawley rats induced colon tumor and colon tumor-associated nonhistone proteins in the chromatin. However, DMH failed to induce colon tumor and tumor-associated nonhistone proteins in Lobund-Wistar rats.