Paclitaxel induces apoptosis in leukemia cells through a JNK activation-dependent pathway.

Paclitaxel (PTX) is a mitotic inhibitor widely used in chemotherapy for many types of cancers, including solid tumors and hematological malignancies. However, the molecular basis of the anti-proliferation activity of PTX is not fully understood. In this paper, we focused on the role of c-Jun N-terminal kinase (JNK) pathways in PTX-induced apoptosis and proliferation inhibition. The effects of PTX were examined in human leukemia cell lines and patients' chronic lymphocytic leukemia (CLL) cells in relation to mitochondrial events, apoptosis, and perturbation of JNK activation using flow cytometry, siRNA, mitochondrial membrane potential determination, and western blotting. Exposure of cells to PTX at concentrations ≥ 10 nM for 18 or 24 h resulted in a significant release of cytochrome c from mitochondria to the cytosol, cleavages of procaspase 3 and poly (ADP-ribose) polymerase (PARP), and JNK activation, leading to apoptosis. The pan-caspase inhibitor BOC-D-FMK blocked the PTX-induced apoptosis but had no effect on cytochrome c release, suggesting that cytochrome c had been released before caspase activation. Moreover, both pharmacological JNK inhibitors SP600125 and JNK siRNA dramatically blocked PTX-induced apoptosis, cytochrome c release, caspase 3, and PARP cleavage. These findings demonstrate that JNK activation plays a critical role in the induction of apoptosis mediated by PTX in human leukemia cell lines and CLL patient-derived primary cancer cells, and this event is upstream of cytochrome c release, caspase 3, and PARP cleavage.

Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

Analysis of Antarctic proteobacteria by PCR fingerprinting and screening for antimicrobial secondary metabolites.

Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa).

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.

Molecular cloning and characterization of a tocopherol cyclase gene from Lactuca sativa (Asteraceae).

Tocopherol cyclase is a rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding tocopherol cyclase (designated as LsTC) was cloned from lettuce (Lactuca sativa) for the first time by rapid amplification of cDNA ends (RACE) and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTC was 1675 bp, with an open reading frame of 1521 bp, encoding a tocopherol cyclase protein of 506 amino acids, with a calculated molecular mass of 56.76 kD and an isoelectric point of 6.49. Comparative analysis revealed that LsTC has a close similarity with tocopherol cyclases from other plant species. Bioinformatic analysis indicated that LsTC shares a common evolutionary origin based on sequence and has the closest relationship to tocopherol cyclase from Helianthus annuus. Quantitative RT-PCR analysis suggested that expression of LsTC is induced and strengthened by oxidative stresses, such as strong light and drought. This cloning and characterization of LsTC will be helpful for further understanding of its role in the tocopherol biosynthesis pathway and provide a candidate gene for metabolic engineering of vitamin E.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide.

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes.

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F-->I, 21/21) and 212 (G-->S, 19/21) or (G-->N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G-->D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L-->F) and 129 (M-->I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W-->C), and 5/21 specimens showed another novel change at codon 115 (G-->A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.