The Effects Of Iron Deprivation On MDR Of Leukemia Cell During TPA-induced K562 Cell Differentiation

Multidrug resistance (MDR) of leukemic cell is one of major factors leading to therapy failure and relapse. mdrl -coded P-gP overexpression is primary mechanism responsive for MDR. Reversing or decreasing MDR is one of marjor strategies for MDR. Iron is essential to normal cellular activity. Disorder of iron metabolism was document in patients with malignant tumor diseases. Ferritin level and transferritin receptor expression in leukemia cell were higher than that in normal hemacytes. Differentiation induction therapy has become one of an important approaches for clinical treatment of leukemia. MDR was also emerged in the differentiation therapy for leukemia result in resisting the differentiation reagent. This experiment was carried out to provide the experiment evidence for resolving MDR of leukemia in clinical, using K562 cell as a research target, and TPA-induced K562 cell differentiation as an experiment model.[Objective] 1. To observe K562 cell differentiation lineages and to explore the expression of EGRl mRNA, mdrlmRNA, P-gP and the relationship between these expressions with cell differentiation during the process of TPA-induced cell differentiation.2. To explore the relationship between iron state in leukemia cell with the transcription factor EGRl and the MDR of leukemia by interfering iron metabolism of leukemia cell during the process of TPA-induced K562 cell differentiation, trying to find a new mechanism of leukemia MDR on molecular level and look for an effective method to decrease the leukemia MDR[Methods] l.After K562 cell was cultured in vitro, Wright's staining and NSE staining and iron staining were used to observe the differentiation direction of K562 cell and iron level in K562 cell. 2.1mmunohistochemistry was used to detect the P-gP expression. 3. Flow cytometry was applied to test DNR fluorescence intensity to explore its function. 4.RT-PCR was applied to assay the expressions of EGRlmRNA^ mdrlmRNA and H-FnmRNA.[Results] l.Our investigation revealed that the K562 cell morphologicUy differentiated into monocyte/macrophages lineages with TPA treament; Most of NSE-stained K562 cells showed positive, and were inhibited by NaF in experiment group. Most of K562 cells stimulated with TPA arrested at G0+G1 phase of cell cycle. The change of differentiation antigens on the cellular surface CD33 expression was not obvious, while CD 14 was greatly increased over time treated by TPA. EGRl mRNA was expressed in TPA-inducd differentiation cell, while the control group had no expression, the expressions of mdrlmRNA and P-gP were both increasedgradually over a time course, and DNR fluorescence intensity increased during TPA-induced cell differentiation. 2. K562 cell growth was inhibited remarkably in TPA-Fe group, Most cells looked like leukemia blasts in control group and in TPA-Fe group in morphology, but most cells became more mature in K562 + TPA group and TPA+Fe group. Most K562 cells with NSE staining showed strong positive in groups of TPA or TPA+Fe treatment, but negative or weak positive both in control group and TPA-Fe treatment group. Most of K562 cells stimulated with TPA-Fe cell-cycle arrest at S phase, the expression of CD33 differentiation antigens on the cellular surface did not change in four groups, and CD 14 greatly decreased in TPA-Fe group. After iron deprivation, iron-containing granules and H-FnmRNA expression decreased as well as EGRlmRNA, while mdrlmRNA and P-gP, and DNR fluorescence intensity increased in K562 cell.[Conclusion] l.Our experiment indicats that TPA can induce K562 cell to differentiate toward monocyte/macrophage lineages. The expressions of mdrlmRNA, P-gP and its function all gradually increased during TPA-induced differentiation. 2. EGRlmRNA was expressed during the process of TPA-induced K562 cell differentiation, and it may participate in the process of K562 cells differentiate into monocyte/macrophages 3.K562 cell growth was inhibited and tis differentiation was blocked at S phase, so the iron deprivation agent could be regarded as a supplementary anticancer drug in leukemia chemotherapy. Although,with some blocking effect of cell differentiation, it needs further studies to establish the roles of iron deprivation agent can be used in leukemic induced-differentiation therapy. EGRlmRNA expression was reduced after iron deprivation, so it mayparticipate in blocking TPA-induced K562 cell differentiation course. 4. Iron deprivation reduced EGRlmRNA expression as well as MDR of K562 cell. Our results show for the first time that lower expression of MDR may relate to the decreasement of H-ferritin and transcriptional factor EGR1 expression after iron deprivation in K562 cell.