| Tumor gene and tumor suppressor genes are hotspots in the research field of leukemogenesis. It's believed that the origin of leukemia was the results of abnormal proliferation, differentiation arrest and inhibition of apoptosis in the leukemic stem or progenitors. Numerous oncogenes are found to be implicated in these processes. Although more and more the marker oncogenes and the related chromosome aberrations are reported in acute myeloid leukemia such as AML1/ETO in t(8;21) positive AML-M2a or PML/RARa in t(15;17) AML-M3 cases, there're even more oncogenes with transcription factor activity have been investigated for their roles in the pathogenesis of leukemia.Gfi-1 is a zinc finger transcription factor which was first identified as a target for proviral insertion activation in a rat lymphoma cell, an interleukin-2 dependent cell line; Activation of Gfi-1 rendered the cells growth factor independence. Abnormal expression of Gfi-1 was also detected in neurospongioma, breast cancer and endometrical cancer. Recent researches revealed that Gfi-1 was a very important transcription factor in the signal transduction pathway in the regulation of cell growth and cell cycle. All these studies indicated that Gfi-1 may act as a tumour gene and may play key roles in the cancer pathogenesis.Acute myeloid leukemia is a clonal disease with different kinds of chromosome aberrations and gene mutations; we sought to investigate the expression of Gfi-1 in AML samples and the clinical relevance. Using RT-PCR, we examined transcription expression of Gfi-1 in AML cells from patients in different stages and analyzed the relationship of Gfi-1 expression and biological characteristics of AML. Western blot and confocal microscopy were also used to detect Gfi-1 expression in protein level and the intracellular sub-localization of Gfi-1. The same experiments were also applied to CD34+ cells sorted from mononuclear cells of healthy or AML patients. To further explore the possibility of targeting the Gfi-1 as anti-cancer therapy, we treated HL-60 leukemic cells with different concentrations of Adriamycin (ADM) and observed the cell growth, differentiation and apoptosis of the cells; at the same time, the change of Gfi-1 and other apoptosis proteins such as Bax and Bcl-2 were analyzed by western blot and RT-PCR.Part one The expression of oncogene Gfi-1 in acute myeloid leukemiaObjective to study the expression of oncogene Gfi-1 in acute myeloid leukemia and the clinical relevance.Methods 1. The expression of Gfi-1 in 5 leukemic cell lines(HL-60, Jurkat, K562,U937, Molt-4 ) and AML92(M1 17, M2 30, M3 16, M4 4, M5 21 and M6 4) were detected by RT-PCR and Western-blot. The patients with de novo AML 73,complete remission(AML -CR)19. 10 benign diseases (including patients with iron deficiency anemia, hemolytic anemia and idiopathic thrombocytopenic purpura ) and 5 healthly adults served as normal control.2. Correlation between positive expression rate of Gfi-1gene and clinical parameters ( age, gender, splenohepatomegaly and peripheral white blood cells count) was analyzed in 92 patients with AML, the patients based on peripheral white blood cells count were divided into two groups: 1. mild tumor burden: peripheral blood WBC <20×109 /L; 2. high tumor burden: peripheral blood WBC≥20×109 /L.3. The expression and sub-location of Gfi-1 protein were observed in bone marrow mononuclear cell with AML and cell lines by confocal microscopy.Results 1. Gfi-1mRNA in 5 leukemic cell lines could be detected by RT-PCR, but intensity of expression was different. Strong positive expression of Gfi-1 gene in HL-60, K562 and Jurkat cell lines were detected(R≥1.0), medium intensity expression of Gfi-1 gene in U937, Molt-4cell lines were detected(0.60.05), but related with peripheral white blood cells count. the positive expression rate of Gfi-1 mRNA of peripheral blood WBC≥20×109 /L in BMMNC with AML was 100%( 44/44); the positive expression rate of Gfi-1mRNA of peripheral blood WBC < 20×109 /L in BMMNC with AML was 75.86%(22/29), their difference had statistical significance (P<0.01). The difference of expression rate of Gfi-1mRNA in normal control and patients with peripheral blood WBC<20×109 /L had statistical significance (P<0.05).3. The expression of Gfi-1 protein in AML the expression of Gfi-1 protein in patients with AML was analyzed by Western-blot. The expression of Gfi-1 protein(2.15±0.23) in patients with de novo AML was obviously higher than control(0.46±0.02)(p<0.01). The Gfi-1 protein expression in patients with de novo AML was higher than AML-CR(0.78±0.01 ) (p<0.01). The Gfi-1 protein expression in patients with AML-CR was higher than controls, but their contrast was not different in statistics.4. The sub-location of Gfi-1in cells by Confocal microscopy.Gfi-1 could be detected by Confocal in 5 leukemic cell lines, but intensity of expression was different. In HL-60, K562 and Jurkat cell lines expressed strong positive Gfi-1mRNA, the expression of Gfi-1 flourescence was strong positive expressed in nucleus and cytoplasm. In U937, Molt-4 cell lines expressed moderate positive Gfi-1mRNA, Gfi-1 flourescence was moderate positive expressed in nucleus and cytoplasm. The expression of Gfi-1 in BMMNC with 13 AML and 5 healthly adults was analyzed that Gfi-1 was located at nucleolus, specifically, located at initial nucleolus in AML. Strong intensity Gfi-1 expression in the most nucleolus in AML was observed and medium intensity in cytoplasm. Gfi-1 expression in normal BM and other hematonosis was detected a little or not.Conclusion The expression of Gfi-1 protein and mRNA in acute myeloid leukemia was detected. Its expression was significant increased in high tumor burden. Through analysis association in the expression of Gfi-1 and clinical parameters, we found that the high expression of Gfi-1 was related with peripheral blood WBC, and not related with the age, gender, splenohepatomegalia in patients with AML. We proposed that Gfi-1 could be an oncogene in regulation of cell growth in myeloid leukemia, and serves as an independent influence factor in the onset of AML.Part two The expression of oncogene Gfi-1 in normal and leukemic hemopoietic progenitor cellObjective to study the expression of oncogene Gfi-1 in normal and leukemic hemopoietic progenitor cell.Methods BMMNC from 16 patients diagnosed with de novo AML (M1 7,M2 6,M3 3), 3 AML patients in CR and 3 healthy adults were obtained using lymphocyte isolation medium. Then CD34+ cells were sorted from BMMNC with FCM. The expression of oncogene Gfi-1 was detected with RT-PCR and Western-blot in CD34+ fraction.Results 1.through RT-PCR, the expression of Gfi-1 mRNA in AML and AML-CR was 1.42±0.13 and 0.58±0.04, which all were higher than normal level(0.14±0.02) p<0.01 and p<0.05). Compared with AML and AML-CR, the expression of Gfi-1 mRNA was different, the expression of Gfi-1 mRNA in AML was higher than AML-CR (p<0.05). The expression of Gfi-1 mRNA were compared between each subsets in AML, them were no difference in statistics (p>0.05).2. through Western-blot, the expression of Gfi-1 protein in AML and AML-CR was 1.26±0.14 and 0.65±0.04, which all were higher than normal level(0.24±0.02) (p<0.01 and p<0.05). Compared with AML and AML-CR, the expression of Gfi-1 mRNA was different, the expression of Gfi-1 protein in AML was higher than AML-CR (p<0.05). The expression of Gfi-1 protein were compared between each subsets in AML, them were no difference in statistics (p>0.05).Conclusion the expression of Gfi-1 of CD34+ cells in healthy adults were detected, the expression of Gfi-1 protein and mRNA also were detected in CD34+ cells of AML and AML-CR, and the expression of Gfi-1 was significantly increased in patients with de novo AML. It indicated that Gfi-1 maybe oncogene of hemopoietic progenitor cell or tumor stem cell growth in AML. Part three Study of apoptosis inducing effect of Adriamycin on HL-60 cells and its relationship with Gfi-1 and the relevant apoptotic geneObjective the study was to explore the effect of Adriamycin (ADM) on HL-60 cells in vitro and the mechanism of expression changes of relevant apoptotic genes and oncogene Gfi-1.Methods HL-60 cells at logarithmic phase was added ADM in different density (0mg/L,0.1 mg/L,0.2 mg/L,0.5 mg/L,1.0 mg/L,2.0 mg/L,5.0mg/L). After 24h, 48h and 72h, interfered HL-60 cells were analyzed using following methods. The cell proliferation of HL-60 cells resulted from ADM was detected through MTT tests. The apoptosis cells were stained by Hoechst33258 (8g/L), and the change of morphocytology of apoptosis cells were observed through fluorescent microscope. The apoptosis rate was essayed by flow cytometry (FCM) using Annexin-V/PI staining , and ruptured DNA was essayed by the DNA electrophoresis; the expression changes of Gfi-1,Bcl-2,Bax mRNA and protein were examined by RT-PCR and Western-blot after HL-60 cells were treated with different concentrations of ADM for 24 hours.Results the suppressed growth in HL-60 cells had related with dose and time of treatment by ADM. HL-60 cells showed gradually high apoptosis in ADM of different density, along with the increasing ADM, typical Apoptosis morphology appeared in 2.0 mg/L ADM, including capacity of cells growing down, cell membrane wrinkling and curling, vacuole in cytoplasm, karyopyknosis, chromatin tightly closed and agglomerated and was distributed around caryotheca. But HL-60 cells died in 5.0 mg/L ADM. The percentage of apoptosis cells had related with dose of treatment after ADM, along with the increasing ADM, apoptosis rate of HL-60 cells showed gradually up to cell necrosis, the greatest apoptosis rate was 22.75±1.92 in 2.0 mg/L ADM, but apoptosis rate of HL-60 cells were descended to 7.43±0.56 in 5.0 mg/L ADM. Genome DNA of HL-60 cells emerged typical electrophoresis lane in 2.0 mg/L ADM. When concentration was 0.5 and 2.0 mg/L ADM, with RT-PCR essay, Gfi-1mRNA and Apoptosis correlated gene Bax and Bcl-2mRNA had different changes in 0mg/L, 0.5 mg/L and 2.0mg/L ADM, strap of Gfi-1 expression was obscure gradually, relative value of gray scale was p <0.05, had significant discrepancy through statistics analysis. This result illustrated that the expression of Gfi-1 mRNA was decreased gradually when the concentration of ADM was increased gradually. Strap of Bax expression was clear gradually, relative value of gray scale was p <0.01, had significant difference through statistics analysis. This result illustrated that the expression of Bax mRNA was increased gradually when the concentration of ADM was increased gradually; the expression of Gfi-1 mRNA and Bax mRNA was negative correlation. The change of straps of Bcl-2 mRNA was not obvious, the relative value of gray scale was p >0. 05, had no significant difference for statistics analysis, this result indicated that there was not significant change of Bcl-2 mRNA between 0.5 and 2.0 mg/L ADM. The results of Gfi-1 protein and Bax and Bcl-2 protein in HL-60 cells treated by different ADM were similar with the expression of their mRNA through Western blot.Conclusion ADM can inhibit the proliferation of HL-60 cells and induce apoptosis of cells. The mechanism that ADM induced apoptosis on HL-60 cells maybe related to the low expression of Gfi-1 and activated the expression of Bax.
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