The Interaction Between PTEN,NOTCH1and MYC Gene In The Pathogenesis Of T-ALL Cell JURKAT

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is a tumor ofmalignant clone of T lymphocytes, the incidence rate is about10-15%inchildhood ALL and for adult is about25%. The pathogenesis of T-ALL ofteninvolves genetically changes in hematopoietic precursor cells, leading to thedisorder of cell cycle regulation, blocked maturation, self-replication,excessive proliferation, and inhibition of apoptosis. In recent clinicaltreatment, the complete remission rate and long-term survival of T-ALLpatients have been significantly improved, but still30%of patients exist thephenomenon of recurrence and poor prognosis. Therefore, further study ofT-ALL pathogenesis and explore new therapeutic targets are still of greatsignificance. PTEN is the first discovered well-known tumor suppressor genewith a dual specificity phosphatase activity. It functions as a lipid phosphataseto dephosphorylate PIP3into PIP2. As a second messenger, PIP3promotescell proliferation, inhibits apoptosis through the activation of the PI3K/AKTpathway. And PTEN become the most important negative regulator of theanti-apoptotic signal transduction pathway PI3K/AKT by reducing the amountof PIP3, reversing the role of PI3K, which lead to a series of signalingmolecules downstream changes so that calles cell cycles arrest in G1phase,inhibit tumor cell growth and induce apoptosis. The present study show thatboth solid tumors and malignant tumors of the hematopoietic system, thereexist lack of PTEN function. In recent years, people also pay attention to therole of PTEN in T-ALL, but the exact mechanism is unclear. NOTCHsignaling pathway affected multiple processes, such as proliferation,differentiation and apoptosis of hematopoietic cells. In T cell development,NOTCH1may play an important role, especially in the tuning of variousstages of T cell development. In recent research, NOTCH1mutations and abnormal activation become hot topics, which may be a new targeted therapyin treating T-ALL. PTEN and NOTCH1are closely related with theoccurrence and development of T-ALL, but the specific mechanism andinteraction in the disease need to be further elucidated. NOTCH1mutationsare common in T-ALL, which lead to over-activation of NOTCH signalingpathway, promote T cells growth, block the development of B cells, and leadto the formation of T cell lymphoma. Activated NOTCH signal transductionpathway leads to tumor formation mainly from the following two aspects:1.Induce over-expression of proto-oncogene C-MYC;2. Persistent activation ofP13K/AKT and other anti-apoptotic pathways. But the study about thefrequent problem of GSI(γ-secretase inhibitor),a NOTCH signaling pathwayinhibitor, drug resistance found that PTEN gene deletion may be the mainproblem. If there exists mutual regulatory mechanism between NOTCH1andPTEN in tumor occurrence and development needs further study. While c-mycis an important downstream molecule of the NOTCH signaling pathway, butalso as an oncogene over expressed in many tumors. Reduce or block itsoverexpression can inhibit tumor cell growth and induce apoptosis. But howto trigger T-ALL development is not yet clear. But may be used as a bridge toconnect the PTEN/AKT and NOTCH1, and provide a targeted treatment forleukemia. This experiment aimed to study the influence of cell proliferationand apoptosis of PTEN gene on T-ALL cell lines JURKAT, and try to initiallyinvestigate its mechanism, detect the expression changes of T-ALL relatedgenes(NOTCH1and C-MYC) after PTEN gene was transfected, and withfurther study of the interaction between PTEN and C-MYC in thedevelopment of leukemia can provide an experimental basis to cure leukemia.Methods: The recombinant adenovirus containing green fluorescentprotein (GFP) gene and PTEN gene (Ad-PTEN-GFP) or empty vector(Ad-GFP) was transfected into JURKAT cells, the cell apoptosis wereassessed by flow cytometry (FCM). The cell proliferation were determined byMTT. The mRNA expression level and difference of PTEN, MYC, andNOTCH1were detected by Reverse transcription PCR (RT-PCR) method. Results:1This experiment successfully constructed the PTEN gene high expressionvector by using adenovirus which carried wild-type PTEN gene and greenfluorescent protein (GFP) gene transfect JURKAT cell lines.2After transfected with adenovirus in JURKAT cell line for48h, cell RNAwas extracted RNA, RT-PCR showed that PTEN gene was highlyexpressed in JURKA cells, indicating successful transfection.3JURKAT cells which were transfected with wild-type PTEN geneshowed proliferation were inhibited, compared with the empty vectortransfected and untransfected group was statistically significant (P <0.05),but between the latter two groups there were no significant differences (P>0.05).4The enhanced apoptosis rate were observed in JURKAT cells transfectedwith wild-type PTEN, but the same changes had not been observed inJURKAT cells transfected with empty vector and untransfected JURKAT(P＜0.05).5After transfection for48h, RT-PCR results showed NOTCH1and MYCmRNA expression levels were significantly lower than those transfectedwith empty vector group and untransfected group (P <0.05), and nodifference in expression levels between the later two groups (P>0.05).Conclusions:1This experiment successfully constructed the PTEN gene high expressionvector by using adenovirus which carried wild-type PTEN gene and greenfluorescent protein (GFP) gene transfect JURKAT cell lines, significantlyinhibited the human T-lymphoblastic leukemia cell lines JURKAT cellproliferation, promoted apoptosis. This indicates that PTEN can inhibitT-ALL tumor cells growth and induce apoptosis by inhibit PI3K/AKTpathway.2The PTEN gene which is high expressed, down-regulates not onlyNOTCH1expression level but also C-MYC, and their down regulation hascertain correlation. This indicates that PTEN/PI3K/AKT singling pathway can regulate NOTCH1singling pathway, which is related with C-MYCgene regulation.