| Backgroud and ObjectiveAcute leukemia is a clonal malignant blood diseases of hematopoietic stem cell. The resistance of acute leukemic cells to chemotherapeutic drug and disease relapse are the main reasons leading to treatment failure. Study focus on the main mechanisms of drug resistance are the most important field in clinical research on acute leukemia. DNA repair abnormalities of leukemia cells and abnormal pharmocogenomic expression are closely involved in tumor resistance to chemotherapeutic agents. Therefore, discovery of these molecular markers are helpful in designing retional clinical chemotherapeutic regimens, with increased effective and optimal individual therapy. Among these genes, ribonucleotide reductase subunit M1 (RRM1) is an important one. RRM1 is one subunit of ribonucleotide reductase (RR)., Its expression was related to gemcitabine resistance in non-small cell lung cancer and pancreatic cancer and other solid tumors. So, RRM1 plays an important role in the prognosis of patients. The study of RRM1 mainly focused on solid tumors and few in leukemia. RRM1 gene expression is upregulated in refractory acute myeloid leukemia by gene array technology, and the significances are still open to explore. In present study, RRM1 mRNA expression in bone marrow mononuclear cells from patient with acute leukemia was detected by RT-PCR, and the correlation beween the expression level of RRM1 and cell apoptosis induced by cytarabine and gemcitabine in vitro was evaluated. The relationship between the expression level of RRM1 mRNA and clinical efficacy was analyzed after following up of patients.Material and methods1. Research object:Total 38 patients with acute leukemia were enrolled for the research, who hospitalized in the Department of Hematology of the First Affliated Hopsital of ZhengZhou Univesrity from March to June,2009. The diagnosis of acute leukemia is made based on complete blood count, bone marrow morphology, cytochemistry, cytogenetic and immunophenotype, according to the criteria for diagnosis of acute leukemia. These patients have a mean age of 36 years old (ranged from 16 to 63 years old), with 20 males and 18 females.22 cases are AML (7 cases of M2,8 cases of M3,2 cases of M4,5 cases of M5) and 16 case ALL (9 cases of B-ALL, 7 cases of T-ALL). Ten patients with non-malignant hematological diseases were set up as control. These patients have a mean age of 38 years old (ranged from 21to 57 years old), with 4 males and 6 females.2. RT-PCR:Bone marrow mononuclear cells were separated, total RNAs were extracted by TRIZOL. cDNA were reverse transcripted, and PCR were performed in the primers of the RRM1 mRNA. The products of PCR were electrophoresed in agarose gel, the bands were visualized by UV light and the optic density of each band was measured. The ratio of RRM1 mRNA/β-action mRNA was defined as the expression level of RRM1 mRNA.3. Assessment of apoptosis:Bone marrow mononuclear cells were treated with Ara-C and Gemcitabine at 37℃,5%CO2 and saturated moisture for 24h, and the cellular apoptosis was assessed using flow cytometry.4. Patients follow-up:The clinical response of patients to chemotherapy and overall survival were observed by following up patients.5. Statistical analysis:The data were analyzed using Sotfware SPSS16.0. Numeric data were expressed as mean±standard deviative (x±s) or Median(M).The measurement data was analyzed byχ2-test. Paired means were analyzed by Independent-Samples t test, and multiple means were analyzed by ANOVA. Kaplan-Meier was used for survival analysis, with Log-rank test for significance.The standard of singificant level was a=0.05.Result1.Expression of RRM1 mRNA in acute leukemiaRRM1 mRNA was expressed in 22 out of 38 cases (57.9%) of de novo acute leukemia, two out of ten cases (20%) of non-malignant hematological diseases expressed RRM1 mRNA. The difference of RRM1 mRNA expression between two groups were statistically significant (P<0.05).2. Expression of RRM1 mRNA between subtypes of acute leukemia.RRMl mRNA was expressed in 13 out of 22cases (50.9%) of AML. The positive rate was 56.3% in 16 cases of ALL. There was no statistical significance between AML and ALL (P>0.05).3. The relationship between the RRMl mRNA expression levels and drug-induced apoptosis.After bone marrow mononuclear cells from patients with acute leukemia were treated with Ara-C and gemcitabine for 24 hours, cell apoptosis were detected, which were significantly higher than control group. After cells treated with Ara-C, apoptosis (8.07%±0.97%) that expressed high level of RRMl mRNA were significantly lower than those (31.97%±3.14%) with low expression of RRM1 mRNA (P<0.01). After cells treated with gemcitabine, apoptosis were (8.32±1.14)% in patients with high expression of RRM1 mRNA, as compared with (33.58±4.19)% in patients with low expression of RRM1 mRNA(P<0.01).4. Expression of RRM1mRNA with patients'clinical features.There is no significant difference between the expression of RRM1 mRNA and patients gender, age, blasts in bone marrow and extramedullary infiltration (P>0.05).5. The relationship between the expression of RRM1 mRNA and clinical responseIn all the acute leukemia except AML-M3, the median survival time in patients with high expression of RRM1 mRNA was shorter than the patients with low expression of RRM1 mRNA. There was significant difference between them (P <0.05). As for AML-M3 patients, the median survival time was similar in patients with different RRM1 mRNA expression levels.Conclusions1. The expression of RRM1 mRNA in acute leukemia is higher than that in control cases.2. There is no statistical significance of RRM1 mRNA expression between AML and ALL.3. The cells with high expression of RRM1 mRNA have poor apoptosis induced by cytarabine and gemcitabine.4. The median survival with high expression of RRM1 mRNA is shorter than patients with low expression of RRM1 mRNA.
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