Identification of microRNA-205 as a potential prognostic indicator for human glioma.

Altered microRNA-205 (miR-205) expression has been found in glioma tissue samples and cell lines; however, the clinical significance of this is unclear. The aim of this study was to confirm the miR-205 expression pattern in human glioma and to investigate its clinical relevance. Quantitative reverse-transcription polymerase chain reaction assays showed that miR-205 expression was significantly lower in glioma tissues than in non-neoplastic brain tissues (P<0.001). Statistical analysis revealed a significant correlation between low miR-205 expression and both high grade glioma (World Health Organization [WHO] criteria, P=0.008) and a low Karnofsky performance status score (P=0.02). Survival analysis demonstrated that the cumulative 5-year overall survival rate of patients with glioma in the high miR-205 expression group was significantly higher than that in the low miR-205 expression group (P<0.001). Multivariate Cox regression analysis further indicated that miR-205 expression (P=0.01) and WHO grade (P=0.01) were independent prognostic indicators of the overall survival of patients with glioma. Moreover, subgroup analyses revealed that the cumulative 5-year overall survival rate of patients with high grade (III-IV) glioma was significantly worse for the low miR-205 expression group than for the high miR-205 expression group (P<0.001), but no significant difference was found for patients with low grade (I-II) glioma (P=0.09). In conclusion, down-regulation of miR-205 was associated with glioma progression. Our data are the first to suggest that miR-205 holds potential as a prognostic factor for glioma, especially for patients with advanced disease.

Pharmacokinetic, tissue distribution and excretion of ginsenoside-Rd in rodents.

Ginsenoside-Rd (GS-Rd) is one of the major active components of Panax ginseng, and was shown to have the protective effects against several insults. However, we still lack some basic knowledge of GS-Rd, including its pharmacokinetic, tissue distribution and excretion in vivo in experimental animal, such as mice and rats. In this study, HPLC and radioactive tracer assays were performed to determine pharmacokinetic, tissue distribution and excretion of GS-Rd in rodents. After intravascular administration with 20, 50 or 150 mg/kg GS-Rd, the dynamic changes of GS-Rd concentrations in plasma were consistent with a two-compartment model while the concentration of ³H-labeled GS-Rd was rapidly reached the peak in plasma, and distributed to various tissues, among which the highest concentration was observed in the lung.

Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells.

OBJECTIVE: To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. METHODS: 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. RESULTS: NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P < 0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. CONCLUSIONS: The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

[Identification of protein peroxiredoxin 2 related to crystalline NiS-induced neoplastic transformation].

OBJECTIVE: To provide evidence for illustrating the molecular mechanism of nickel carcinogenesis, and to identify the differential expression of protein in crystalline NiS-induced neoplastic transformation of human bronchial epithelial cell by proteomics technology. METHODS: Two dimensional electrophoresis (2-DE) and the ImageMaster 3.10 software were used to analyze the differential expression of protein, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify protein peroxiredoxin 2 (PDX2) related to malignant transformation. RESULTS: The good 2-DE pattern including resolution and reproducibility was obtained. Nearly 700 expressed proteins per 2-D gel were isolated with molecular weights (MW) ranging from 14,400 to 94,000 KD and pI 3 - 10. A protein PDX2 with MW 21,890 KD, pI 5.66, which was highly expressed in malignantly transformed cell, was identified using MALDI-TOF-MS. CONCLUSION: PDX2 was involved in malignant transformation of human bronchial epithelial cell induced by crystalline nickel sulfide.

[Telomerase SiRNA inhibits KB cell growth in human oral squamous cell carcinoma].

OBJECTIVE: To test the telomerase SiRNA on telomerase mRNA and on KB cell growth of oral squamous cell carcinoma. METHODS: We synthesized 21-nucleotide SiRNA duplexes with symmetric 2-nucleotide 3' overhangs corresponding to the target sequence (2 657 approximately 2 675 nucleotide downstream of the start codon) of telomerase mRNA. Telomerase activity, cell proliferation, cell cycle and apoptosis were measured after transfection. RESULTS: Twenty one-nucleotide small interfering RNA (SiRNA) duplexes specifically suppressed expression of endogenous telomerase mRNA in human oral squamous carcinoma KB cells. This inhibitory effect lasted only for about 48 h after transfection. Telomerase activity reduction corresponded to the mRNA suppression. Cell proliferation decreased by 30% at 48 h after transfection and lasted for 120 h after treatment. This inhibitory effect resulted from the block of G(1) to S transition. Apoptosis was not involved in this process. CONCLUSIONS: SiRNA is a powerful tool for studying gene function and can be used as gene-specific therapeutics.

[Crystalline nickel sulfide-induced genomic instability in transformed human broncho-epithelial cells].

OBJECTIVE: To detect the genomic instability in the 16 human broncho-epithelial (16HBE) cells induced by crystalline nickel sulfide so as to provide the scientific basis for further study of nickel-induced cancer molecular mechanism. METHODS: To analyse the genomic instability in transformed 16HBE cells induced by crystalline nickel sulfide by random amplified polymorphic DNA (RAPD). RESULTS: All the 7 random primers selected could amplify 1 - 6 clear PCR bands. There were no significant differences between transformed 16HBE cells and negative control cells in the 4th, 5th, and 7th primers, but in the rest 4 primers there were significant differences, with special PCR bands for the same primer, indicating that genomic instability in transformed 16 HBE cells was induced by crystalline nickel sulfide. CONCLUSION: Crystalline nickel sulfide could induce genomic instability in 16HBE cells.

[A study on the role of DNA repair gene O6-methylguanine-DNA methyltransferase in the development of human lung cancer].

OBJECTIVE: To study the role of O(6)-methylguanine-DNA methyltransferase (hMGMT) in the development of human lung cancer. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) method was applied to measure hMGMT mRNA expression in 150 lung cancer specimens, 40 normal lung tissues, and in the peripheral mononuclear blood cells from 50 lung cancer cases and 50 normal controls. The protein expressions of p53, C-MYC and K-RAS were assessed by immuno-histochemistry. The effects of some exposure factors on the expression of hMGMT gene were analyzed. The relationships between hMGMT gene and cancer related genes p53, C-MYC and K-RAS were investigated. RESULTS: The mRNA of hMGMT was low or absent in 49 of 150 (32.7%) lung cancer specimens, whereas 2 of 40 (5%) normal lung tissues had reduced the levels of hMGMT mRNA. The low expression of hMGMT seemed to be a risk factor of lung cancer, with a OR of 9.22 (2.05-57.65). Reduced expression levels of hMGMT mRNA were observed in 10 of 50 (20%) lung cancer patients' peripheral mononuclear blood cells, and 2 of 50 (4%) blood cells among normal controls. When investigating the exposure factors which affecting the expression of hMGMT gene, we noticed that smoking was suppressing the expression of hMGMT gene. Interestingly, over-expression of K-RAS oncogene was significantly correlated with low expression of hMGMT (P < 0.05). However, the expressions of p53 and C-myc were not correlated with the status of hMGMT gene. CONCLUSION: hMGMT might play an important role in the development of human lung cancer. Low expression of hMGMT gene seemed to be a risk factor for lung cancer which could be used as a valuable biomarker on susceptibility of human lung cancers.

[Alteration of FHIT gene and p16 gene in malignant transformed cells induced by crystalline nickel sulfide].

OBJECTIVE: To detect the alteration of fragile histidine triad (FHIT) gene and p16 gene during malignant transformation of immortal human bronchial epithelial cell line (16HBE) induced by crystalline nickel sulfide, and study the molecular mechanism of nickel carcinogenesis. METHODS: Malignant transformed cells and tumorigenic cells were examined for the alteration of FHIT gene and p16 gene by RT-PCR, DNA sequencing and silver staining PCR-SSCP. RESULTS: Compared with those of control 16HBE, neither mutation of exon2 or exon2-3, abnormal expression in p16 gene nor mutation of FHIT exon5, 6, 7 and 8, exon1-4 or exon5-9 were observed in transformed cells and tumorigenic cells. But aberrant transcript or FHIT gene expression loss were observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in FHIT gene, the deletion of exon6, exon7 and exon8 and an insertion of 36 bp sequence replacing exon6-8, was confirmed by sequencing. CONCLUSION: FHIT gene, not p16 gene, could play a definite role in nickel carcinogenesis. Alterations of FHIT gene induced by crystalline NiS could be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation, and FHIT gene could be one of the important target genes activated by exotic carcinogens.

[Study on lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by carcinogen].

OBJECTIVE: To investigate lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by chemical carcinogens with cDNA microarray. METHODS: The gene expression patterns were detected in all specimens by cDNA microarray which representing 4 096 different human genes. The differences in gene expression among 6 cases of human lung squamous cell carcinoma tissues and 6 normal lung tissues were analyzed. The different gene expression patterns between the normal human bronchial epithelial cell lines (16HBE) and the malignant transformation of human bronchial epithelial cells induced by Benzo(a)pyrene metabolite BPDE (anti-Benzo(a)pyrene diol-epoxide,BPDE) and crystalline nickel sulfide were also studied by that method. The similar changed genes among those gene expression patterns were identified as lung carcinogenesis associated genes. RESULTS: Among the 4096 genes of cDNA microarrays, there were 171 genes expressed differently among lung cancer tissues and normal lungs, 143 genes expressed differently between BPDE transformed cells and normal 16HBE cell lines, 151 genes differed between nickel sulfide transformed cells and normal 16HBE cell lines. By comparing the gene expression profiles, there were 89 similar changed genes which might be associated with human lung carcinogenesis, 39 of which were up regulated: 6 oncogenes, 4 cell cycle control genes, 6 cell proliferation genes, 8 metastasis genes, 3 neuroendocrine genes, 1 drug-resister gene, 1 anti-apoptosis gene, 1 oxidative gene and other 9 genes. 50 genes were down-regulated: 7 tumor suppression genes, 11 DNA repair genes, 1 antioxidant genes, 3 GST family genes, 3 cell framework genes, 2 apoptosis induced genes, 5 signal conduction genes, 5 cytokines and their receptor genes, 7 metabolization genes, 1 cell matrix genes, and other 5 genes. CONCLUSION: cDNA microarray can be applied to study gene expression profiles effectively and to screen human lung carcinogenesis associated genes.

[Effect of aberrant DNA methylation on the expression of cancer-related genes in Cd-transformed cells].

OBJECTIVE: To study aberrant DNA methylation potentially resulting in changes in the expression of cancer-related genes as a possible epigenetic mechanism for cadmium carcinogenesis. METHODS: Genomic DNA isolated from CdCl(2)-transformed BALB/c-3T3 cells was digested with Mse1 (methylation non-sensitive) alone or with Mse1 and BstU1 (methylation sensitive). The resulting DNA was analyzed for aberrant methylation using PCR-based technique-Methylation-Sensitive Restriction Fingerprinting (MSRF). Several DNA fragments differentially methylated in the transformed cells identified by MSRF were confirmed by Southern hybridization analysis using the aberrantly methylated DNA fragments as the probes. RESULTS: Aberrant DNA methylation was identified in the transformed cells. DNA sequencing and sequence similarity analysis identified one of the aberrantly methylated DNA fragments as the p16 tumor suppressor gene. CONCLUSION: DNA hypermethylation is known to result in gene silencing, it appears that hypermethylation of p16 gene may represent a possible epigenetic mechanism for Cd-induced cell transformation and carcinogenesis.

Blocking the translation elongation factor-1 delta with its antisense mRNA results in a significant reversal of its oncogenic potential.

In spite of the strong evidence for the carcinogenic activity of cadmium and its related compounds, the underlying molecular mechanisms that lead to malignant transformation in cells exposed to cadmium remain unknown. Recently, Joseph et al. [J. Biol. Chem. 227:6131-6136, 2002] have identified, cloned, and characterized the mouse Translation Elongation Factor-1 delta sub-unit (TEF-1 delta, GenBank Accession Number AF304351) as a novel cadmium-responsive proto-oncogene. Presently, additional studies regarding the oncogenic potential of TEF-1 delta have been carried out. Transfection of NIH3T3 cells with the pcDNA3.1 expression vector containing the TEF-1 delta cDNA in the sense (5'-->3') orientation resulted in overexpression of the encoded 31 kDa protein. Transfection-mediated overexpression of TEF-1 delta protein resulted in transformation of the cells as evidenced from the appearance of transformed foci. Cotransfection of the cells with a mixture of plasmid DNA consisting of TEF-1 delta cDNA in the sense (5'-->3') and in the antisense (3'-->5') orientation resulted in significant inhibition of translation of the TEF-1 delta protein. Antisense TEF-1 delta mRNA-mediated inhibition of translation of TEF-1 delta protein, furthermore, resulted in inhibition of TEF-1 delta-mediated transformation of NIH3T3 cells as evidenced from the decrease in the number of transformed foci. These results further confirm that overexpression of TEF-1 delta is oncogenic and the antisense TEF-1 delta mRNA expression reverses its oncogenic potential.