[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.

[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.

[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.