Study Of The Expression Of TGF-β Receptors In Leukemia Cells

Leukemia is a hematopoietic stem cell malignant clonal disease, which is characterated with leukemia cell over-proliferation, blocked differentiation and apoptosis. In physiological conditions, hematopoietic is strictly regulated by a variety of positive and negative cytokines, which is maintaining the stability of blood micro-environment. As one of the major negative factors, TGF-β₁ plays an important role in the regulation of proliferation, differentiation and apoptosis of blood cells. In the classically described signaling pathway, TGF-β₁ acts through a complex of TGF-βRI and TGF-βRâ…¡transmembrane receptors to induce phosphorylation of Smad proteins, leading to their accumulation in the nucleus to regulate target gene expression.Dysfunction of TGF-β₁ and its signal transduction pathway may result in over-proliferation, blocked differentiation and apoptosis.In our previous study, we found that the expression of TGF-β₁ was lower in serum of patients with leukemia and leukemia cells than health donors. We firstly found that leukemia cells had two isoforms of TGF-βRâ…¡, named TGF-βRâ…¡A and TGF-βRâ…¡B (exon2 75 base paire deletions). The recombinant pEFIRES-N-TGF-βRâ…¡A and pEFIRES-P -TGF-βRâ…¡B were transfected into K562 cell line which has low expression of TGF-βRâ…¡mRNA .Using lipofectamine 2000, K562 cells stable transfected with TGF-βRâ…¡A and TGF-βRâ…¡B were selected with puromycine and neomycine, named TGF-βRâ…¡A-K562 cell and TGF-βRâ…¡B-K562 cell.The effects of TGF-β₁ on the proliferation,differentiation and apotosis ofTGF-βRâ…¡A-K562 cells and TGF-βRâ…¡B-K562 cells were detected by MTT and FCM. The results showed that 1 ng/ml TGF-β₁ could inhibit the proliferation of TGF-βRâ…¡A-K562 cells, induced the differentiation and apotosis.However, the same dose of TGF-β₁ neither inhibited the proliferation of TGF-βRâ…¡B-K562 cells, nor induced the differentiation and apoptosis of TGF-βRâ…¡B -K562 cells. While 2ng/ml TGF-β₁ could also inhibit the proliferation of TGF-βRâ…¡B-K562 cells, but it was not significant as TGF-βRâ…¡A K562 cells. It has not been reported whether it was different on TGF-β₁ binding with the two isforms of TGF-βRâ…¡. Neither the expression of TGF-βRâ… and the two isforms of TGF-βRâ…¡in leukemia cells has been reported. In this study,TGF-βRâ…¡and its isoforms were detected by absolute quantification RT-PCR, the expression and the coding sequence of TGF-βRI in acute leukemia cells were also detected by RT-PCR and DNA sequencing in leukemia cells. And the affinity of TGF-β₁ with TGF-βRâ…¡A and TGF-βRâ…¡B in K562 cells was detected by radiation binding assay. Further,5 cases of acute leukemia primary cells, which have different expression level of TGF-βRI and TGF-βRâ…¡,were cultured to observe inhibitation by adding different doses of TGF-β₁.Results:1. The expression of TGF-βRâ…¡A and TGF-βRâ…¡B mRNA were lower in acute leukemia cells than that in normal bone marrow mononuclear cells (P=0.0057, 0.0006); the expression of TGF-βRâ…¡B mRNA were lower in chronic leukemia cells than that in normal bone marrow mononuclear cells (P=0.0002), but the expression of TGF-βRâ…¡A mRNA was not significantly different between chronic leukemia cells and normal bone marrow mononuclear cells (P=0.1215).2. RT-PCR showed that the expression level of TGF-βRI mRNA in acute leukemia cells and normal bone marrow mononuclear cells depend on individuals, there was no significant difference between the two groups. No deletion and mutation were observed in TGF-βRI mRNA coding sequence.3. There was no significant difference on the affinity of TGF-β₁(P=0.8876)between TGF-βRâ…¡A K562 cells and TGF-βRâ…¡B -K562 cells.4. 1ng/ml TGF-β₁ could not inhibit the proliferation of 5 cases acute leukemia primary cells(P>0.05); 5ng/ml TGF-β₁ could inhibit the proliferation of 3 cases acute leukemia primary cells which has higher expression of TGF-βRI and TGF-βRâ…¡ (P<0.05),but the same dose TGF-β₁ could not inhibit the 2 cases acute leukemia primary cells which has lower expression of TGF-βRI and TGF-βRâ…¡(P>0.05); 25 ng/ml and 50ng/ml TGF-β₁ could inhibit the proliferation of all the 5 cases acute leukemia primary cells(P<0.05).Conclusion:1. The expression of TGF-βRâ…¡A and TGF-βRâ…¡B mRNA in acute leukemia cells were lower than that in normal bone marrow mononuclear cells; however, in chronic leukemia cells,only TGF-βRâ…¡B mRNA were lower than that in normal bone marrow mononuclear cells.2. There was no significant difference on the affinity of TGF-β₁ between TGF-βRâ…¡A and TGF-βRâ…¡B.3. TGF-β₁ could inhibit the proliferation of acute leukemia primary cells, the effects depend on the dosage of TGF-β₁ and the expression levels of TGF-βreceptors. The proliferation was inhibited more obviously in leukemia cells with higher expression of TGF-βreceptors compared with those with lower expression of TGF-βreceptors at the same dosage of TGF-β₁; and the proliferation was inhibited more obviously with the increasing of the dosage of TGF-β₁ in the same case.