[Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the role of antigen-processing machinery (APM) component defects in HLA class I antigen down-regulation in laryngeal squamous cell carcinoma (SCC) and to assess the clinical significance of these defects. METHODS: Fifty-one formalin-fixed, paraffin-embedded SCC specimens were examined for the expressions of APM component transporter associated with antigen processing (TAP1) and low molecular weight polypeptide (LMP-7) and HLA class I antigen by immunohistochemistry. RESULTS: HLA class I antigens, TAP-1 and LMP-7 expressions were down-regulated in 56.9% (29/51), 39.2% (20/51) and 45.1% (23/51) of the tested specimens respectively, whereas HLA class I antigens, TAP-1 and LMP-7 expressions lost in 21.6% (11/51), 33.3% (17/51) and 27.5% (14/51) of the tested specimens respectively. TAP-1 and LMP-7 expressions were significantly correlated with HLA class I antigen expression (r=0.460, P<0.05 and r=0.685, P<0.05, respectively). HLA class I antigens down-regulation was significantly correlated with T stage (χ2=8.61, P<0.05). Both TAP-1 and LMP-7 down-regulations were significantly correlated with T stage (χ2 values were 9.72 and 8.97 respectively, P<0.05) and TNM stage (χ2 values were 9.18 and 7.70 respectively, P<0.05). TAP-1, LMP-7 and HLA class I antigen down-regulations were significantly associated with reduced patients' overall survival (P<0.05) and disease-free survival (P<0.05). Multivariate analysis showed lymph node metastasis, recurrence and HLA class I antigen down-regulation were unfavorable prognostic factors (P<0.05). CONCLUSIONS: Down-regulated expressions of HLA class I antigen and APM component TAP-1 and LMP-7 occur frequently in laryngeal squamous cell carcinoma, by which cancer cells could avoid immune surveillance, while HLA class I antigen down-regulation is a major contributing factor to tumour progression and mortality.

[Advances in human papillomavirus therapeutic vaccine].

Human papillomavirus (HPV) infection is very common but with limited therapies available. Although the prophylactic vaccination will be promoted worldwide soon, it can only show its benefits decades later. For individuals who already have established infections and dysplasias, it has little efficacy. In contrast, the therapeutic vaccines bridge the temporal deficit by fighting against the established HPV infections and HPV-related diseases. HPV oncogenes may be delivered in viral and bacterial vectors, in peptides or protein, in nucleic acid form, or in cell-based vaccines. This review summarizes the clinical trials of HPV therapeutic vaccines under the way and the different preclinical research strategies that are under investigations.

Neuroprotective effect of lysophosphatidic acid on AbetaP31-35-induced apoptosis in cultured cortical neurons / 生理学报

It has been reported that lysophosphatidic acid (LPA) at its lower concentrations prevents apoptosis induced by serum-deprivation in cultured cortical neurons when LPA is added into the cultural medium with serum withdrawal. The present study was designed to investigate whether LPA could also block the apoptosis induced by beta-amyloid peptide fragment 31-35 (AbetaP31-35) in cultured cortical neurons by using techniques of DNA fragmentation electrophoresis, HO33342 staining, and TUNEL examinations. The results showed that pretreatment of LPA suppressed the AbetaP31-35-induced apoptosis only when LPA was applied to the cultured neurons with lower concentrations (1-10 micromol/L) and especially, with a preceding time of 12-24 h before the AbetaP31-35 exposure. These facts imply that LPA also acts as a neuroprotective factor against AbetaP31-35-induced apoptosis, though the mechanism underlying the protective action in this case may be more complex than that involved in the serum deprivation-induced apoptosis.

[Isolation of E1A-related drug-sensitive new genes by suppression subtractive hybridization].

BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH). METHODS: To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times. CONCLUSION: Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.

[Expression of HPV16-E6 and E7 oncoproteins in squamous cell carcinoma tissues of esophageal cancer and non-cancer tissues].

BACKGROUND & OBJECTIVE: Evidences indicate that high-risk type human papillomavirus (HPV) are closely associated with the carcinogenesis, progression and transformation of several kinds of human tumors. This study was designed to determine the expression of HPV16- E6 and E7 oncoproteins in normal tissues, dysplasia tissues, and carcinoma tissues of patients with esophageal cancer and to investigate the biological significance of high-risk type HPV in the esophageal squamous cell carcinogenesis. METHODS: HPV16-E6 and E7 oncoproteins were determined using immunohistochemical staining in normal mucosa tissues (70 cases), dysplasia tissues (43 cases), and carcinoma tissues (18 cases). RESULTS: The positive rates of HPV16-E6 in the tissues of normal mucosa, dysplasia, and carcinoma of esophagus patients were 59.3%,88.4%,and 83.3%,respectively; the positive rates of HPV16-E7 protein were 62.1%, 90.7%, and 88.9%, respectively. The positive rates of HPV16-E6 and E7 in dysplasia and carcinoma of esophagus were significantly higher than those in normal mucosa (P< 0.05). Double expression of HPV16-E6 and E7 in normal mucosa was 25.7%, while in dysplasia and carcinoma were 88.3% and 83.3%,respectively. CONCLUSION: HPV16-E6 and E7 are highly associated with esophageal squamous cell carcinogenesis. And cooperation of HPV16-E6 and E7 may play an important role in genesis of esophageal squamous carcinoma.

Dual action of lysophosphatidic acid in cultured cortical neurons: survival and apoptogenic / 生理学报

The effect of lysophosphatidic acid (LPA), with a wide range of its different concentrations, upon cultured mouse cortical neurons was assessed by electrophoresis of DNA fragments, HO33342 and TUNEL stainings, and also by ultrastructural examination at times. The results showed that administration of LPA at lower concentrations (0.1-30 micromol/L) dose-dependently protected cortical neurons from apoptosis that was induced by deprivation of serum from the cultural medium, while 50 micromol/L or higher concentrations of LPA failed to show this effect; and moreover, the concentrations higher than 50 micromol/L induced apoptosis in neurons cultured in serum-containing complete medium. These results suggest that a moderate concentration of LPA may play as a survival factor in apoptotic cortical neurons, while an excessive level of LPA induces apoptosis in neurons cultured in complete medium.

[Effect of E1A gene on in vitro growth inhibition and radiochemosensitivity of lymph node metastasis cells of human head and neck squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Adenovirus type 5 early region 1A (E1A) gene has been found to be a tumor suppression gene recently. The protein of E1A gene regulates the expression of many cellular genes positively or negatively, and possesses the activities of inducing differentiation of tumor cell, reversing of malignant phenotype, anti-carcinogenesis and anti-metastasis. Study of E1A protein on the treatment of lymph node metastasis of human head and neck squamous cell carcinoma (HNSCC) was not reported. This study was designed to investigate the growth inhibition and radiochemosensitivity of E1A gene on human lymph node metastasis cell line 686LN-1 derived from the patient with human tongue squamous cell carcinoma in vitro and its mechanism. METHODS: The pcDNA3-E1A recombinant plasmid, designed for high-level expression of E1A gene in a variety of eukaryotic cell lines,was transfected into 686LN-1 cells mediated by lipofectamine. To observe the growth inhibition of E1A gene on the cells, the growth curve and doubling time were investigated. Cells before and after transfection were treated with cisplatin, paclitaxel, bleomycin, and 5-fluorouracil (5-FU) for 24 hours or irradiation, respectively, then the changes of sensitivity were tested by MTT assay. The redistributions of cell cycle were analyzed by flow cytometry. Immunocytochemical staining was used to detect the expression of p53 and HER-2/neu. RESULTS: Compared with the vector-transfected cells (686LN-1-vect cells), the E1A-transfected cells (686LN-1-E1A cells) grew slowly, and the doubling time elongated (1.41-fold). 686LN-1-E1A cells showed distinct sensitivity to the anticancer drugs and irradiation. According to the IC(50) value, the sensitivity of 686LN-1-E1A cells increased approximately 8-fold to cisplatin, 20-fold to bleomycin, 10-fold to paclitaxel, 1-fold to irradiation compared with 686LN-1-vect cells. However, the sensitivity to 5-FU did not change. The cell cycle was dramatically arrested at G(2)/M phase in the 686LN-1-E1A cells. E1A gene remarkably suppressed the expression of HER-2/neu gene in 686LN-1-E1A cells. CONCLUSION: E1A gene can significantly inhibit the growth rate of lymph node metastasis cell line 686LN-1 of HNSCC. Moreover, it also slightly enhance the cell sensitivity to antitumor drugs and irradiation. These functions of E1A gene may be associated with its ability to suppress the HER-2/neu expression and to arrest the cell at G(2)/M phase.

[Selective myelo-protection by MDR1 and MnSOD genes regulated by a specific promoter].

OBJECTIVE: To study the specific protection of myeloid cells from chemotherapeutic agents and radiation. METHODS: The recombinant retroviral vectors containing MDR1 gene and MnSOD gene regulated by APN myeloid promoter were constructed and introduced into myeloblastic cell line KG1a and hepatoma cell line BEL7402. The resistance of the cells to antitumor drugs and radiation were analyzed by cell survival assay. In vivo, the murine bone marrow cells were isolated and infected by the retroviral particles, which were transplanted into recipient mouse treated with paclitaxel or X-ray. The murine white blood cell (WBC) was counted in order to assay the effects of MDR1 or MnSOD gene on hematopoiesis in the course of chemotherapy and radiotherapy. RESULTS: The resistance to chemotherapeutic agents such as cochicine, Vp-16, vincristine, doxorubcin and paclitaxel were elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in KG1a cell line transduced with MDR1 gene. The resistance to radiation increased 3.7 folds at the dose of 10 Gy compared with parental cells in KGla cell line transduced with MnSOD gene derived by APN promoter. In contrast, the chemosensitivity and the radiosensitivity showed no significant change in BEL 7402 cell line transduced with MDR1 gene and MnSOD gene. In vivo, the WBC counts in the mouse introduced with MDR1 gene or MnSOD gene were higher than those in the control mouse (P < 0.01). CONCLUSION: The expression of MDR1 gene and MnSOD gene regulated by APN myeloid promoter is effective on myelo-specific protection without enhancing the resistance of tumor cells in vitro. The hematopoiesis can be reconstituted in vivo during anticancer drug or radiation treatment. This study may provide experimental evidence and new clues for myeloprotection of cancer patients being treated with chemotherapy and/or radiotherapy.

[Effects of E1A gene on the growth and chemosensitivity of transplantation tumor of nude mice].

OBJECTIVE: To study the effects of E1A gene on the growth and chemosensitivity of transplantation tumor of nude mice of lymph node metastasis cell line (686LN-1) of human tongue squamous cell carcinoma. METHODS: 686LN-1, 686LN-1-vect and 686LN-1-E1A cells were transplanted into nude mice, then the time of tumor formation, growth rates and weight of tumor were observed. To study the effects of the E1A gene on bleomycin sensitivity in vivo, 686LN-1 cells were injected into nude mice. After tumor formed, bleomycin, E1A gene and E1A gene + bleomycin were given respectively as therapy. The tumor volume was calculated, and growth curve was plotted. Representative histological sections were taken from mice bearing transplantation tumor either treated or control groups, and expression of HER-2/neu was detected. RESULTS: In nude mice, the expression of E1A gene significantly suppressed the growth rates and elongated the time of tumor formation. Bleomycin or E1A Gene and E1A gene + bleomycin can suppress the growth rates of transplantation tumor of nude mice, the suppressed rates of tumor growth was 53.13%, 76.83% and 96.65%, respectively. The expression of E1A gene can significantly suppressed the expression of HER-2/neu gene in 686LN-1-E1A cells. CONCLUSION: E1A is able to significantly inhibit the growth rate of transplantation tumor of nude mice of lymph node metastasis cell line (686LN-1) of human tongue squamous cell carcinoma, and dramatically enhance the sensitivity of the cells to cytotoxic agents in vivo. Above of all functions of E1A gene may be related to that it suppressed the expression of HER-2/neu gene.

[MnSOD gene regulated by aminopeptidase N promoter specifically protects bone marrow from radiation].

BACKGROUND & OBJECTIVE: It is an effective way to induce radio-tolerant gene into hematopoietic cells in bone marrow for overcoming the suppression of radiotherapy on hematopoietic system. However, this also increases the radiation tolerance of tumor cells. This study was designed to investigate a method to specifically protect bone marrow cell from being damaged by radiation, along without increasing resistance of tumor cell to radiation. METHODS: The retrovirus vector of manganese superoxide dismutase (MnSOD) gene regulated by aminopeptidase N (APN) bone marrow-specific gene promoter was constructed and induced into myeloblastic KG1a and cancer cell BEL7402. MnSOD mRNA level was analyzed by PT-PCR; MnSOD activity in the cells was determined; the sensitivity of bone marrow cell and hepatic carcinoma cell to x-ray was detected by cell survival test; the cell apoptosis was analyzed with flow cytometry and fractural DNA electrophoresis. RESULTS: The MnSOD mRNA level and enzyme activity in KG1a cells transferred with the gene was obviously increased. Expression of MnSOD mRNA drove by APN myelo-specific promoter effectively inhibited apoptosis of KG1a cells induced by radiation and endowed KG1a cell line with the enhancement of tolerance to radiation, which increased by 3.7 folds compared to parental cells at the dose of 10 Gy. In contrast, the level of MnSOD mRNA, the enyme activity of MnSOD and the radiosensitivity had no significant change in BEL 7402 cells transduced with MnSOD gene. CONCLUSION: APN bone marrow-specific promoter could control MnSOD gene expression highly in myeloid cell and lower in cancer cell. In the process of killing of cancer cell by x-ray, MnSOD gene regulated by APN bone marrow-specific promoter could specifically protect myeloid cell. This study provides a new clue to solve the bone marrow suppression in high dose radiotherapy.