OBJECTIVE: To investigate the effect of eukaryotic translation initiation factor 4E(eIF4E) on the autophagy of CD138 plasma cells in multiple myeloma(MM). METHODS: Multiple myeloma CD138 plasma cells were treated with eIF4E inhibitor 4EGI, the changes of autophagy-related factors LC3-II and Beclin1 were detected by fluorescent quantitative PCR and Western blot, the changes of cell proliferation inhibition were detected by MTT assay, and cell apoptosis was detected by flow cytometry. RESULTS: Quantitative fluorescence PCR showed that after treatment of myeloma cells with 4EGI, the expression levels of LC3-II and Beclin1 mRNA gradually increased with the enhancomer of 4EGI concentration and the prolongation of action time, and the differences were statistically significant (48 h: LC3-Ⅱ,r=0.942, Beclin1,r=0.952; 80 μg/ml: LC3-Ⅱ,r=0.966, Beclin1,r=0.998）; Western blot showed that with the enhancement of 4EGI concentration, the expression of LC3-II and Beclin1 protein gradually increased（LC3-Ⅱ,r=0.923, Beclin1,r=0.977）; CCK-8 showed that the inhibition rate of cells gradually increased （r=0.996）; the apoptotic rate of 4EGI-treated groups (23.23±4.47, 7.59±1.67, 2.03±0.19) was significantly different from that of control group (0.03±0.04) (P<0.05). CONCLUSION The inhibition of eIF4E can activate the autophagy of CD138 plasma cells in multiple myeloma and induce the death of myeloma cells.
Autophagy ; Beclin-1 ; Cell Line, Tumor ; Eukaryotic Initiation Factor-4E ; Humans ; Multiple Myeloma

Few papers have reported that the HIV-1 replication was inhibited by p53 in the infected cells. However, the detail mechanism for the p53-medicated HIV-1 suppression has not yet been clearly demonstrated. In our previous report, we addressed that p53-mediated Tat suppression is very likely associated with PKR. In the present study, we found that the amounts of p53 in the HIV-1 infected cells increased over 10 times in the early stages of infection as much as those in normal cells. Particularly noteworthy is that the both exogenous p53 and endogenous p53 enhanced PKR expression in the transformed or treated cells, and the amounts of PKR induced by p53 were almost equivalent to those induced by interferon. In the PKR promoter studies using Ppkr-CAT (CAT reporter system under the control of PKR promoter), CAT activity induced by p53 was stronger than that by interferon, suggesting that the p53-mediated PKR expression might be more efficient than interferon under the control of PKR promoter. Co-immunoprecipitation experiments showed that PKR directly binds to Tat protein. We established eIF-2alpha dominant negative (S51A) Jurkat cells (JK/eIF2alpha-51A) to block the PKR-mediated cell cycle arrest or apoptosis. In the JK/eIF2alpha-51A cells, not only p53 but also PKR inhibited the Tat activity. Taken together, our results demonstrate that the HIV-1 infection induces p53, which enhances PKR expression by promoter activation, followed by the inhibition of the Tat activity, finally resulting in the inhibition of HIV-1 replication. Detail mechanisms for the PKR-mediated Tat inactivation are under investigation.
Animals ; Apoptosis ; Cats ; Cell Cycle Checkpoints ; Eukaryotic Initiation Factor-2 ; Gene Products, tat ; HIV-1* ; Humans ; Immunoprecipitation ; Interferons ; Jurkat Cells

OBJECTIVETo study the expression of eIF4E, p-eIF4E (Ser 209) and Mcl-1 gene in the pathological scars and to investigate its role and its probable mechanism in the pathogenesis of abnormal scar.

METHODSQuantitative real-time PCR and Western Blot was performed to detect the expression and distribution of mRNA and protein of eIF4E and Mcl-1 in hypertrophic scar (10 cases), keloid (10 cases), normal scar (10 cases), and normal skin (10 cases). Western Blot was performed to detect the expression and distribution of protein of p-eIF4E in hypertrophic scar (10 cases), keloid (10 cases), normal scar (10 cases), and normal skin (10 cases).

RESULTSThe expression of eIF4E mRNA and protein were 1.38 +/- 0.45, 1.23 +/- 0.23 in the normal skin (10 cases); 5.400 +/- 0.450, 5.460 +/- 0.460 in normal scar (10 cases); 0.597 +/- 0.060, 0.590 +/- 0.040 in hypertrophic scar (10 cases) and 0.694 +/- 0.066, 0.697 +/- 0.022 in keloid (10 cases). The expression of p-eIF4E protein in the normal skin (10 cases), normal scar (10 cases), hypertrophic scar (10 cases), and keloid (10 cases) were 0.202 +/- 0.037, 0.216 +/- 0.019, 0.426 +/- 0.026, 0.433 +/- 0.027. The expression of Mcl-1 mRNA and protein were 1.510 +/- 0.660, 1.400 +/- 0.530 in the normal skin (10 cases); 6.65 +/- 0.85, 7.23 +/- 1.53 in normal scar (10 cases); 0.589 +/- 0.059, 0.660 +/- 0.063 in hypertrophic scar (10 cases) and 0.870 +/- 0.118, 0.914 +/- 0.064 in the keloid (10 cases). The positive rate of mRNA and protein of eIF4E and Mcl-1 was not statistically different between the hypertrophic scar and keloid (P > 0.05), while they were all remarkably significant between normal scar and abnormal scar (P < 0.05). The phosphorylation of eIF4E in pathological scar was higher than that in control group. In pathological scar, mRNA and protein of eIF4E and Mcl-1 showed a strong positive correlation.

CONCLUSIONSThe result indicates that the expression of eIF4E, p-eIF4E and Mcl-1 is increased in pathological scar. eIF4E plays an important role in pathological scar. Its activity is regulated by its phosphorylation. Therefore, eIF4E, p-eIF4E and Mcl-1 overexpression may play an important role in the proliferation of fibroblasts and in the pathogenesis of pathological scar.

Adolescent ; Adult ; Case-Control Studies ; Cicatrix ; metabolism ; Eukaryotic Initiation Factor-4E ; genetics ; metabolism ; Female ; Humans ; Keloid ; metabolism ; Male ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; metabolism ; Phosphorylation ; RNA, Messenger ; genetics ; Young Adult

Animals ; Cadherins ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Early Growth Response Protein 1 ; genetics ; metabolism ; Estradiol ; physiology ; Eukaryotic Initiation Factor-3 ; metabolism ; Genes, Tumor Suppressor ; physiology ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Iron ; metabolism ; Neoplasms ; metabolism ; pathology

OBJECTIVE: To explore the mechanism of paclitaxel on the protein expression of human cervical carcinoma cell line HCE1. METHODS: The total proteins extracted from paclitaxel-treated HCE1 cells were analyzed by 2-dimensional gel electrophoresis (2-DE), and compared with those from untreated HCE1 cells. The differential proteins were identified by mass spectrometry. Western blot was used to determine the differential expression levels of the 2 proteins. RESULTS: At 24 hour after paclitaxel (0.05 mumol/L) treatment, 2-DE images of paclitaxel-treated and paclitaxel-untreated cells were analyzed. Forty-two differential proteins were found. Twenty-one differential proteins among 42 proteins were analyzed by mass spectrometry, among which 15 proteins were identified, including peptidyl-prolylisomerases A (PPIase A),alpha-enolase,keratin 8,heat shock protein 90, eukaryotic translation initiation factor 1A, and so on. CONCLUSION Fifteen proteins in human cervical carcinoma cells paclitaxel-treated and paclitaxel-untreated are found by proteomic techniques. These proteins may be involved in the proliferation inhibition of human cervical carcinoma cells by paclitaxel.
Antineoplastic Agents, Phytogenic ; pharmacology ; Biomarkers, Tumor ; analysis ; DNA-Binding Proteins ; analysis ; Eukaryotic Initiation Factor-1 ; analysis ; Female ; Gene Expression Profiling ; Genome ; Humans ; Keratin-8 ; analysis ; Paclitaxel ; pharmacology ; Phosphopyruvate Hydratase ; analysis ; Proteome ; analysis ; Proteomics ; methods ; Tumor Cells, Cultured ; Tumor Suppressor Proteins ; analysis ; Uterine Cervical Neoplasms ; metabolism ; pathology

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.
Antibiotics, Antineoplastic/pharmacology ; Apoptosis/*drug effects ; Apoptotic Protease-Activating Factor 1 ; BH3 Interacting Domain Death Agonist Protein ; Blotting, Western ; Bronchi/metabolism/*pathology ; Carcinogens/*pharmacology ; Carrier Proteins/metabolism ; Caspases/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells/metabolism/*pathology ; Eukaryotic Initiation Factor-4E/metabolism ; Flow Cytometry ; Humans ; Nitrosamines/*pharmacology ; Protein Biosynthesis ; Proteins/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA Cap-Binding Proteins/*physiology ; Sirolimus/pharmacology ; Time Factors ; bcl-2-Associated X Protein