Retinoid Acid Induced Cell Differentiation And Apoptosis Targeting RARα

Objective:Retinoic Acid(RA),the most potent biologically active metabolite of vitamin A,suppresses proliferation by inducing differentiation and apoptosis in a variety of cell types through modifying expression of RA-target genes.The biological effects of RA on cell differentiation of malignant cells are mediated by retinoid receptors,notably retinoic acid receptor alpha(RARα).The recent finding that CAK- RARαdiassociation, me'nage a' troisl(MAT1) degradation and RARαhypophosphorylation in the presence of RA,coordinate cell cycle G1 exit and transition into differentiation in myeloid leukemia.We further study CAK- RARαsignaling events on osteosarcoma cell,human hematopetic stem cell differentiation and acute T lymphoma leukemia cell apoptosis based on this.We first test the inhibition and mecenism of RA on osteosarcoma cells,as well as comfirm the hypothesis that RA-induced loss of RARαphosphorylation mediates differentiation of osteosarcoma cells.We then investigated whether CAK-RARαsignaling coordinates CAK-dependent cell division and transition into differentiation in normal primitive hematopoietic cells.At last we investigated the effects and mechanism of ATRA in mediating antiproliferative responses in T lymphoma Molt3 cells.Methods:1) For proliferation assay,human osteosarcoma cells U2OS and MG63 treated with 5μM ATRA or trandusec with lentiviral- RARαS77A,cell growth curve was performed by counting cells each day using typen blue staining.For cell cycle analysis,cells were stained with PI and analyzed by flow cytometry.Osteoblastic differentiation marker OPN expression was detected by RT-PCR and Western blot.The expressions of CAK-RARαsignaling factors CDK7,p27 and p21 were shown by Western blot.Microarray was used to compare the gene profile of ATRA-treated or lentiviral- RARαS77A transduced U2OS cell.2) For proliferation assay,human hematopoietic stem cell(HSC),common myeloid progenitor cell(CMP) and granucytic myeloid progenitor cell(GMP) treated with 1μM ATRA or trandused with lentiviral- RARαS77A,cell growth curve was performed by counting cells each day using trypan blue staining.For cell differentiation analysis,cells were stained with giemsa and the morphology was dectect under microscope.The expressions of CAK-RARαsignaling factors CDK7,MAT1,RARαand pRb during RA-induced myeloid differentiation in HSC,CMP and GMP cells were shown by immuonfluorence, RT-PCR and Western blot.The interaction of CAK-RARαsignaling factors was detected by immunonprecitation.3) For proliferation assay,human acute T lymphoblastic leukemia cell treated with 5μM ATRA,cell growth curve was performed by counting ceils each day using trypan blue staining.For cell apoptosis assay,cells were stained with PI and analyzed by flow cytometry.The expression of apoptosis related protein caspase-3,PARP,Bcl-2 and BAX were detected by Western blot.The mRNA and protein expressions of CAK-RARαsignaling factors RARαand p21 were shown by RT-PCR and Western blot.Results:1) Here we report that ATRA(5μM) suppresses proliferation in both U2OS and MG63 osteosarcoma cells by inducing G1/G0 phase arrest through trypan blue staining and FACS.By using RT-PCR and Western blot,we found that the expression of mature osteoblastic marker OPN was induced,CAK-RARαsignaling factors CDK7 and p21 were downregulated,and p27 was upregulated in ATRA-treated U2OS cells.By using microarray,the regulator in RARαrelated pathway,osteoblastic differentiation pathway and cytokines were activated with ATRA treatment in U2OS cells.2) Here we report that ATRA(1μM) suppresses proliferation in HSC,CMP and GMP cells by inducing myeloid differentiation through trypan blue staining for growth curve and giemsa staining for morphology change.By using immunoflunrence and Western blot, we found that the expression of CAK- RARαsignaling factors CDK7 and RARαwere increased,MAT1 and p21 were downregulated,and meanwhile the phosphoralate level of CDK7,pRb and RARαwas reduced in ATRA-treated U2OS induced normal myeloid differentiation cells.By using microarray,the regulator in RARαrelated pathway, osteoblastic differentiation pathway and cytokines were activated with ATRA treatment in U2OS cells.By using immunonprecipetation,we found that CAK-RARαdiassociation,MAT1 degradation and RARαhypophosphorylation in the presence of ATRRA,coordinate with myeloid differentiation in hematopoietic stem cells.3) Here we report that lentiviral- RARαS77A suppresses proliferation in both U2OS osteosarcoma cells through trypan blue staining.By using RT-PCR and Western blot,we found that the expression of mature osteoblastic marker OPN was enhanced in RARαS77A transduced cell with ATRA treatment.By using microarray,the 4 regulators in RARαrelated pathway,18 factors in osteoblastic differentiation pathway and 20 cytokines were activated in RARαS77A transduced cell compared with blank cells.4) Here we report that lentiviral-RARαS77A suppresses proliferation in CD34+ cells through trypan blue staining.By giemsa staining,it was shown that RARαS77A enhanced ATRA-induced myeloid differentiation.By using RT-PCR,we found that the expression of p21 was upregulated in RARαS77A transduced cells.5) Here we report that ATRA(5μM) suppresses proliferation in acute T lymphoblastic leukemia Molt3 cells by inducing apoptosis through trypan blue staining and FACS.In our study,a decrease in Bcl-2 and increase in Bax protein expression was observed in Molt3 cells, following the increase of Bax/Bcl-2 ratio,we observed an activation of both caspase 3 and PARP that were cleaved after 24 h treatment with ATRA treatment by using Western blot.Meanwhile it was shown that RARαreduction is involved in upregulating p21 expression in mediating RA-induced Molt3 cell apoptosis.Conclusion:It was concluded that CAK- RARαevents play a pivotal role in ATRA-induced osteosarcoma cell and hematopoietic stem cell differentiation.This regulation is similar to ATRA-induced myeloid differentiation in leukemia cell. Transduced hypophosphoralate RARαcan not only inhibit osteosarcoma and CD34+ cell proliferation but also enhance ATRA-induced differentiation.At last RARαreduction is involved in upregulating p21 expression in mediating RA-induced Molt3 cell apoptosis.