Specific suppression of beta-secretase gene expression by short interfering RNA in SK-N-SH cells.

OBJECTIVE: To test the effect of short interfering RNAs (siRNAs) of beta-site APP cleaving enzyme (BACE) on inhibiting the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR. The PCR products were inserted into the retrovirus plasmid pLXSN. The interfering vector was identified as pLXSN/ EGFP-U6-siBACE. The SK-N-SH cell line was produced, which can highly expressed BACE. The inhibitive effect of BACE siRNA on BACE expression was examined by fluoroscopy and immunohistochemistry tests. RESULTS: The interfering vector, pLXSN/EGFP-U6-siBACE, was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cell and reduced the production of Abeta. CONCLUSION: BACE siRNA inhibits the expression of BACE gene of mammalian, which has implications for RNA interference of Alzheimer's disease.

A phase-I clinical trial of active immunotherapy for acute leukemia using inactivated autologous leukemia cells mixed with IL-2, GM-CSF, and IL-6.

UNLABELLED: We evaluated the efficacy and toxicity of vaccination in 29 patients with relapsed or refractory acute leukemia using inactivated autologous leukemia cells combined with interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6. MHC-I, MHC-II, and B7-1 expression status on the surface of leukemia cells and the cytokine profile of IFN-gamma and IL-10 in serum before and after vaccination was detected. RESULTS: Five achieved a complete remission (CR) and six a partial remission (PR) in this vaccination procedure. Adverse effects were erythema, swelling erosion, and even ulcers at vaccination sites and low grade fever during the first three days of vaccination. No other significant side effects were observed. The expression of MHC-I and MHC-II on leukemia cells was 100% and 90% positive, respectively. B7-1 was exclusively expressed on some cases of M4 and M5. The efficacy of the vaccine was statistically associated with the expression status of B7-1 on leukemia cells (P < 0.01). The serum level of IL-10 reduced significantly in the five patients who achieved complete remission (CR) after vaccination as compared with when they were originally diagnosed (P < 0.01). CONCLUSION: We presented here a promising immunotherapy in the treatment of acute leukemia, especially for F.A.B. M5.

Strategies of antigen-specific T-cell-based immunotherapy for cancer.

The critical role of antigen-specific T-cells in the eradication of cancer has been demonstrated in numerous animal models, while significant challenges need to be conquered before antigen-specific T-cell immunotherapy can achieve true success in clinical practice. These challenges include: (1) weak or nonimmunogenicity of spontaneous tumors, (2) negative immune regulation mechanisms of the host immune system, (3) immune inhibition exerted by tumor cells, (4) physical barrier in solid tumor, and (5) escape or resistance to immune attack by tumor cells. Nonetheless, significant success has been achieved in several clinical trials recently, highlighting the possibility of successful manipulation of the immune system for control and elimination of tumor. We focused our study on summarizing the current knowledge and corresponding strategies for improving autologous cytotoxic T-cell (CTL)-based cancer immunotherapy, which include the following aspects: (1) the selection of tumor antigens for stimulation of CTL, (2) strategies of enhancing maturation and antigen presentation activity of dendritic cells (DC), (3) strategies of activation and maintenance of CTL response, and (4) recruitment of suitable immune effector cells to tumor sites. The successful manipulation of the immune system, based on the more and more detailed knowledge of tumor immunology, may finally reach the goal of "immune surveillance of malignancy."

Construction and identification of Fas-targeting siRNA-expressing plasmid.

OBJECTIVE: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. METHODS: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. RESULTS: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. CONCLUSION: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.

Combination chemotherapy with low-dose cytarabine, homoharringtonine, and granulocyte colony-stimulating factor priming in patients with relapsed or refractory acute myeloid leukemia.

As sensitization of leukemic cells with granulocyte colony-stimulating factor (G-csf) can enhance the cytotoxicity of chemotherapy in acute myeloid leukemia (AML), a pilot study was conducted in order to evaluate the effect of G-csf priming combined with low-dose chemotherapy in patients with relapsed and refractory AML. The regimen, G-HA, consisted of cytarabine 7.5 mg/m2/12 hr by subcutaneous injection, days 1-14, homoharringtonine 1.5 mg/m2/day by intravenous continuous infusion, days 1-14, and G-csf 150 microg/m2/day by subcutaneous injection, days 0-14. Thirty-six AML patients were enrolled, 23 refractory and 13 relapsed. Eighteen patients (50%, 95% confidence interval: 33-67%) achieved complete remission (CR) with a median CR duration of 7.2 months, and two elderly patients continued a regimen of maintenance therapy and remained in remission for 26.3 and 14.1 months, respectively, as of last follow-up. Eight patients (22%) experienced neutropenia (median duration: 6 days; range: 2-22 days). Thirteen of the 36 (36%) developed severe infections. Grade 1-2 nonhematologic toxicities were documented, including nausea and vomiting (20%), liver function abnormality (6%), and heart function abnormality (6%). No central nervous system and kidney toxicity was observed. The G-HA regimen is effective in remission induction for refractory and relapsed AML patients and well tolerated in maintenance therapy in some subgroups of elderly patients. Further studies are necessary to elucidate optimum dose and schedule for this regimen to enhance the treatment efficacy of relapsed or refractory AML patients.

Indoleamine 2, 3-dioxygenase expression in cells of human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia and therapeutic effect of its inhibitor 1-methyl tryptophan.

The objective of this study was to investigate the expression and function of indoleamine 2, 3-dioxygenase (IDO) in leukemia. The IDO expressions in human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) were detected by immunofluorescence staining. Constructed leukemia mouse model was used to observe whether the IDO inhibitor, 1-methyl tryptophan (1-MT), has any effect in treating leukemia. The experimental group were fed with 1-MT solution every day while the mice in control group had no further treatment. The results showed that the average ratios of IDO expression were 29.4 +/- 11.2% in M(5) patients and 24.7 +/- 7.96% in ALL patients respectively. After statistical test, IDO expression level in leukemia cells was significantly higher than that of normal mononuclear cells. The tumor decreased gradually in mice treated with 1-MT. At the terminal point of the experiment (88 days after vaccination), the average survival time in the experimental group was 42.3 days while the mice in control group only lived 15.1 days in average, which difference was statistically significant (P < 0.05). Some of the leukemia mice in the experimental group long-term survived without tumor (more than three months after vaccination). It is concluded that human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) express IDO, and both can be treated by 1-MT in mice.

[Correlations of S100A4 protein expression to invasion and metastasis of non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Metastasis-associated protein S100A4 is overexpressed in many malignant tumor cells; it may play a pivotal role in invasion and metastasis of malignant tumors. This study was to determine the expression of S100A4 in human non-small cell lung cancer (NSCLC), and to investigate its correlations to invasion and metastasis of NSCLC. METHODS: The expression of S100A4 in 41 specimens of NSCLC and 6 specimens of normal lung tissues was detected by SP immunohistochemistry. The correlations of S100A4 to clinicopathologic features of NSCLC were analyzed. RESULTS: The positive rate of S100A4 was significantly higher in NSCLC than in normal lung tissues (70.7% vs. 16.7%, P<0.05), and was significantly higher in adenocarcinoma than in squamous cell carcinoma (90.0% vs. 52.4%, P<0.01). The positive rate of S100A4 was significantly higher in stage III-IV than in stage II and stage I NSCLC (100.0% vs. 66.7% and 30.0%, P<0.01), while there was no obvious difference between the latter 2 groups (P>0.05). The positive rate of S100A4 was significantly higher in NSCLC with lymphatic metastasis than in NSCLC without lymphatic metastasis (90.0% vs. 52.4%, P<0.01), and significantly higher in NSCLC with tumor size of > or = 3 cm than in NSCLC with tumor size of < 3 cm (91.3% vs. 44.4%, P<0.001). The expression of S100A4 was closely related to lymphatic metastasis (r=0.480, P=0.001), and tumor size (r=0.288, P=0.017). No significant correlation was found between the expression of S100A4 and pathologic grade of NSCLC (P>0.05). CONCLUSION: S100A4 expression is up-regulated in NSCLC, and closely related to lymphatic metastasis, TNM stage and tumor size, which suggest an important role of S100A4 in the invasion and metastasis of NSCLC.

[Immunological screening for multiple myeloma-associated antigens and their bioinformatics analysis].

This study was aimed to screen the cell cDNA expression library of multiple myeloma HMy2 (MM HMy2) by using "serological analysis of cDNA expression library (SEREX)" technique. The obtained 30 positive clones were all sequenced, and analyzed by BLAST (basic local alignment search tool). The results indicated that 6 known genes and 12 new MM-associated genes were obtained, part of which sequences were spliced by EST (expressed sequence tag) splicing. 6 known genes such as for ring finger protein 167, KLF10, TPT1 protein, p02 protein, cDNA FLJ46859 fis, DNMT1 methyltrasferase etc. have been demonstrated a certain relationship with other tumor's formation, progress and prognosis. The structures and functions of the new genes preliminarily analyzed and predicted by means of bioinformatics showed that MMSA-3, MMSA-8 and MMSA-11 encoding 215, 160 and 122 amino acid residues respectively had the full open reading frames (ORF). All the new genes might be located at euchromosomes but MMSA-1 at sex chromosome. MMSA-4 was highly similar to the protein controlling the transcription of tumor antigen, MMSA-5 might take part in cell phagocytosis, MMSA-7 might inactivated NF-kappaB, and MMSA-12 might be a lymphocytic cytoplasmic protein. The specificity of new genes such as MMSA-3 and MMSA-7 were higher, by a preliminary analysis using CrELISA. It is concluded that tumor antigens screened by this study can be used for early immunological diagnosis, surveillance of minor residual foci, assessment of prognosis, and preparation of tumor vaccine and so on.

[Construction of FAS-targeted RNAi-retroviral vector and its identification for biological activity].

This study was aimed to construct mouse Fas-targeting si RNA-expressing recombinant retroviral vector in order to explore the therapeutic potential of Fas inhibition by siRNA in the treatment of aplastic anemia and to provide a basia for extensive development of RNA interference techninque. The U6(+) 27 promoter cassette and siFas sequence were obtained by PCR method. The U6-siFas fragment was cloned into the multiple restriction site of pLXSN-EGFP and directly downstream of EGFP gene. The resultant retroviral vector pLXSN/EGFP-siFas was packaged using PA317 cell line and tittered using NIH3T3 cell line. P815 cells were infected by the retroviral vector. EGFP expression in P815 was observed under fluorescent microscope and Fas inhibition effect was detected by immunohistochemistry. The results indicated that successfully constructed retrovirus vector pLXSN/EGFP-siFas was could not only deliver siRNA into mammalian cells efficiently and inhibit Fas expression in P815 cells, but also could express EGFP as marker and neomycine resistance gene to allow antibiotic selection. It is concluded that the successful construction of this retroviral vector would greatly facilitate the application of RNA interference and lay the foundation for therapeutic study of Fas inhibition in the treatment of aplastic anemia.