The Basic And Clinical Study Of Notch Signaling In The Acute Leukemogenesis

[Objects]Notch signaling is one of evolutionarily conserved signaling pathway that mediates cell-cell interactions required for a variety of cell-fate decisions during development,it is involved in a variety of cell differentiation,proliferation and apoptosis that affect the development and functions of many organ.Members of the Notch family play critical roles in the determination of cell fates and during embryogenesis they also play crucial developmental roles.Mammalian Notch1 was discovered because of its involvement in(7; 9) chromosomal translocation seen in human T cell acute lymphoblastic leukemia(T-ALL), and the aberrant Notch signaling was subsequently shown that it may contribute to tumorigenesis.During the past years,The majority study of Notch signaling pathway was highlighted in the notch1 mutation in the T cell acute lymphoblastic leukemogenesis.Two absolutely opposite view points regarding the function of Notch as Oncogene or Tumor suppressor in B-cell acute lymphoblastic leukemogenesis and acute myeloid leukemogenesis has emerged.In present study,we aim to detect the expression of Notch and its related molecules in bone marrow cells of acute leukemia patients to investigate the possible role of Notch signaling in human leukemogenesis.2 Examine the effects of the Notch ligand Delta1 on the growth and suppression of normal cells and primary leukemia cells in vitro.3 Study the mechanism of Notch ligand Delta1's effects on the leukemia cell lines THP1 and SHI-1 in vitro.We hope to gain deeper insight into the role of Notch pathway in leukemogenesis and provide new opportunity of individual treatment strategy for AL patients in the future.[Methods][Section 1]Chapter 1 The expression of Notch1 and Jagged1 were detected in BM of 118 acute leukemia patients at diagnosis confirmed by MICM and 11 healthy donors by the RT-PCR methods,these patients included 21 T-ALLs,32 B-ALLs AND 65 AMLs, Normalized gene expression level such as notch1 was determined as a ratio between notch1 andβ2—actin.Chapter 2,3 and 4 Real-time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR) was established for detecting notch signaling molecules including Notch1,Notch2,Jagged1,Delta1,Hes1 and GAPDH expression levels in BM including 15 T-ALLs,16 B-ALLs and 18 AMLs at diagnosis(the blast plus prolymphocyte over eighty percent) and 11 donors by ABI PRISM 7700 PCR.Normalized gene expression level such as notch1 was determined as a ratio between notch1 and GAPDH times 10~6.[Section 2]Chapter1 The recombinant Notch ligand Delta1 protein and Human IgG F(c)(as control) were immobilized in 96-well culture plates.We examined the effects of Delta1 on the growth of cells including BM CD34~+ cells of the healthy donors,the cord blood CD34~+ cells of consented donors,the BM CD34~+ leukemia cells of JMML patient, PB CD3~+ cells of the donors and also PB CD20~-CD56~+ cells of the donors by the MTT methods respectively.Chapter 2 the Delta1 protein and Human IgG F(c)(as control) were immobilized as above,We examined the effects of Delta1 on the growth of leukemia cell lines including NB4,K562,THP1,SHI-1 and MO7E by the MTT methods respectively.[Section 3]In chapter 1,in section 2 chapater2,we have found that the growth of two leukemia cell lines THP1 and SHI-1 were suppressed by the Delta1 protein using the MTT method.Subsequently to inhibit of Notch signaling,aγ-secretase inhibitor DAPT was used to examine whether DAPT could rescue the suppression by the Delta1 on the growth of leukemia cell lines THP1 using MTT method.We also performed the following experiment to examine the proliferation and differentiation of the THP-1 cell indued by Delta1 protein. Cell count analysis,cell morphological analysis,NBT reduction,the expression of CD13,CD14 and CD11b,and binding of AnnexinⅤtested by FCM.In the meantime,the expression of Notch signaling targeting gene Hes1 was detected by real -time RT-PCR method.In chapter 2,We performed the same experiments to check the apoptosis and differentiation of the SHI-1 cell by the Delta1 protein.In addition,we also looked at the effect of DAPT on Delta1 induced growth.[Results][Section 1]In chapter 1 The expression of Notch1 and Jagged1 could be detected in all samples of T-ALL,B-ALL,AML and normal donors by the RT-PCR method.The expression levels of Notch1 in T-ALL was significantly higher than that in control group, while the expression levels of Jagged1 in T-ALL was statistically lower than that in control group.The expression levels of Notch1 was no statistical differences between the B-ALL group and the healthy donor group,while the expression levels of Jagged1 in B-ALL was statistically lower than that in control group.No statistical differences coule be found between the AML group and the donors group,wherease the expression levels of Jagged1 in AML was also significantly lower than that in control group.In chapter 2,3 and 4,the results in ALs compared to that in control group determined by the real-time RT-PCR method,The Mean expression levels of Notch1 in T-ALLs at diagnosis were statistically higher accompanied with a lower level of jagged1 in comparesinon with that in control group.However,the Mean expression levels of Notch2,Delta1 and Hes1 were found no differences between the T-ALL group and the control group.The Mean expression levels of Delta1 in B-ALLs at diagnosis were markedly higher than that in control group;While the Mean expression levels of Jagged1 in B-ALLs at diagnosis significantly decreased than those in control group.Again,the Mean expression levels of Notch1,Notch2 and Hes1 were no statistical differences between the B-ALL group and the control group.The Mean expression levels of Jagged1 and Hes1 in AML at diagnosis were statistically decreased than those in control group.While,the Mean expression levels of Notch1,Notch2 and Delta1 were not different between the AML group and the control group.These result concluded by the RT-PCP method in chapter was in concordance with the result concluded by the real-time RT-PCR method in chapter 2,3 and chapter4.[Section 2]In chapter 1,Determined the MTT methods,The Notch ligand Delta1 in the cell culture could not promote or suppress the growth of the hematopoietic cells including BM CD34~+ cells of the donors,the cord blood CD34~+ cells of the donors,the BM CD34~+ leukemic cells of JMML patient,the PB CD3~+ cells of the donors and the PB CD20~-CD56~+ cells from the donors.In chapter 2,On terms of leukemia cell lines failed to Notch ligand Delta1 promote or inhibit the growth of NB4,K562 and MO7E while remarkably suppressed the growth of two monocytic leukemia cell lines,namely THP1, SHI-1 by the MTT methods respectively.[Section 3]In chapter1,Delta1 strongly suppressed the growth of THP1 cells while theγ-secretase inhibitor DAPT could not converse this effect.Delta1 strongly suppressed the proliferation and viability of THP1 cells after treatment with Delta1 for 72 hours. Delta1 partially induced THP1 cells differentiation evidenced by an increased NBT reduction rate to(60±8)%after exposure to Delta1 for 48 hours while the NBT reduction rate in control was(19±5)%.The expression of CD11b and CD14 on the THP1 cells were markedly upregulated after being exposure to Delta1 for 72 hours determined by FCM,suggesting that the THP1 cells were partially induced to differentiation towards monocytic lineage.The apoptoic rate of the THP1 cells treated with Delta1 72 hours late were decreased from(10.2±5.4)%of Human IgG F(c) control group to(6.6±3.7)%of Delta1 group respectively determined by FCM,suggesting that Delta1 did not cause apoptosis of the THP1 cells.The Notch signaling targeting gene Hes1 in the THP1 cells after treatment with Delta1 for 72 hours was upregulated determined by real-time RT-PCR.In chapter 2,Delta1 strongly inhibit the growth of SHI-1 cells and theγ-secretase inhibitor DAPT could partially reverse this effect.Delta1 strongly suppressed the proliferation and viability of SHI-1 cells after treatment of Delta1 for 72 hours.Besides, Delta1 enhanced the apoptosis of the SHI-1.The NBT reduction rates of the SHI-1cell line treated with Delta1 after 48 hours were(17.6±5.2)%while the NBT reduction rates of control group of SHI-1 cells were(15.2±4.6)%,demonstrating no statistical difference between these two groups.The expression of CD13,CD11b and CD14 on the SHI-1cells treated with Delta1 showed no statistical difference to the control group treated with Human IgG F(c),either in preasence or absence of DAPT.The apoptoic rate of the SHI-1 cells treated with Delta1 after 72 hours was markedly upregulated to(39.1±6.7) %,suggesting that Delta1 could exert the apoptotic function on the SHI-1 cells.The treatment of DAPT for 72 hours,decreased the apoptoic rate of the SHI-1 cells to (21.0±2.8)%,implying that DAPT could partially reverse the apoptoic function of Delta1 on the SHI-1 cells.The Notch signaling targeting gene Hes1 in the SHI-1 cells was upregulated for 10 folds(134.5±23.7 vs 1375.1±612.1) by exposure to Delta1 for 72 hours determined by real-time RT-PCR,while this effect was decreased from (1126.5±402.2) to(375.1±121.3) in presence of DAPT.[Conclusion](1) The aberrant Notch signaling was found in BM cells of T-ALL,B-ALL and AML.(2) Notch1 expression levels in T-ALL were strikingly higher than in healthy donors while Jagged1 in T-ALL is evidently lower than in donors,suggesting that abberant Notch signaling may play a role during the acute T cell lymphoblast leukemogenesis;Delta1 expression levels in B-ALL were markedly higher than in donors while Jagged1 in B-ALL is evidently lower than in donors,suggesting that abberant Notch signaling may also play an important role in development of the acute B cell lymphoblast leukemia;While Jagged1 and Hes1 expression levels in AML were strikingly lower than in donors,implying that abberant Notch signaling may be one of the primary cause during the acute myeloid leukemogenesis.(3) The aberrant Notch signaling in the acute leukemogenesis was not only correlated with abnormal expression levels of Notch1 receptor,but also with the abnormal expression levels of ligand Jagged1 and Delta1.(4) Delta1 could suppress the proliferation of the leukemia cell lines THP1 and SHI-1. but was unable to inhibit the growth of the leukemia cell lines NB4,MO7E,K562 and 8266,or to induce apoptosis.Besides Delta1 showed no effect on the BM CD34~+ cells.(5) Delta1 inhibted the proliferation of THP1 cells and induced the differentiation of THP1 cells along monocytic lineage,theγ-secretase inhibitor DAPT could not reverse this effect on the THP1 cells induced by Delta1.(6) Delta1 enhanced the apoptosis of SHI-1 cells,and the apoptotic function of Delta1 on SHI-1 cells could be partially inhibited by DAPT.In summary:The aberrant Notch signaling could be found in T-ALL,B-ALL and AML.The expression pattern of Notch receptors,ligands and target genes were different in various subtypes of acute leukemia,suggesting that the difference expression pattern of molecules related to Notch signaling may confer to subtypes of the acute leukemia in during leukemogenesis.Delta1 suppressed the growth of both monocytic leukemia cell lines THP1 and SHI-1 by either induce monocytic differentiation in THP1 or promoting apoptosis in SHI-1.