The Effect Of Rap1GAP On The Biological Characteristics Of Acute Myeloid Leukemic Cell Lines And Its Possible Mechansim

【Objects】Rap1GAP belongs to GTPase-activating proteins （GAPs） family and plays animportant role during the GDP-GTP cycles of Rap1.Guanine nucleotide exchange factors（GEFs） promote the release of GDP, rendering Rap1to rebind a new GTP to stimulate thedownstream signal cascade. On the contrary, GAPs stimulate the low intrinsic GTPaseactivity of Rap1to hydrolyze GTP resulting in the inactivation of Rap1. Rap1belonging tothe Ras family regulates two important cellular processes: B-RAF/MEK/ERK activationand integrin-mediated cell adhesion and migration. Many studies show Rap1as well as itsupsteam regulators play an important role in the development of human maligancies.Recently, some studies showed that the expression of Rap1GAP was down-regulated inpancreatic cancer, melanoma and thyroid cancer and up-regulated expression of Rap1GAPcan suppress tumor cells proliferation and influence cell invasion ability. All these data ledto the proposal that Rap1GAP may act as a putative tumor suppressor gene in these tumors.The previous data of our laboratory had shown that Rap1GAP may play importantfunctions in Myelodysplastic syndromes （MDS）. Here we want to transfect the leukemiccells using lentivirus to study the function of Rap1GAP in the pathogenesis of acutemyeloid leulemia （AML）.【Methods】[Section1] Quantitative RT-PCR method was used to detect the mRNA level of Rap1GAPin the bone marrow mononuclear cells （BMNCs） of AML and non-malignant blooddiseases patients. The mRNA and protein levels of Rap1GAP in leukemic cell linesincluding HL-60、NB4、SHI-1and U937were also detected. Rap1GAP cDNA wassubcloned into the lentivirus vector using a pair of primers containing BamHI digestion sites. Virus particles were packaged in human293T cells by calcium acid phosphatecoprecipitation method. Human leukemic cells HL-60and NB4were transfected withlentivirus and sorted by FCM using YFP as signal, then the stable monoclonal cellstransfected with Rap1GAP （H1、H2and N1、N2） were obtained by limited dilution method.MTT assay and colony forming assay were used to detect the effect of Rap1GAP on theproliferation ability of leukemic cells. After ARTA or TPA inducted24h、48h and72h,NBT reduction assay and CD11b Mean fluorescence index （MFI） dectection wereundertaked to evaluate the effect of Rap1GAP on cell differentiation. As₂O₃was used toinduce apoptosis in HL-60and NB4cells to study the function of Rap1GAP on cellsurvival ability. Transwell chamber system and gelatin zymography were used to detect theeffect of Rap1GAP on cell invasion ability in vitro.[Section2] All4-week-old BALB/c nu/nu mice were pre-treated by splenectomy, cytoxanintraperitoneal injection, and sublethal irradiation （SCI nu/nu mice）. Then these mice weredivided into five groups at random including the control group （n=5）, the HL-60transplanted group, the MOCK transplanted group, the Rap1GAP-transfected H1, H2groups （n=8for each group）. Except for the control group which were infused with0.2mlIMDM, the other groups were transplanted intravenously with1.2×10⁷different cellssuspended in0.2ml IMDM. After cell inoculation, the nu/nu mice were bred in a laminarflow cabinet for60days. Mice were dissected when they were moribund or deadimmediately. Bone marrow slides of nu/nu mice were stained by Wright’s to observe theinfiltration of leukemia cells. The pathological section of different organs was done byroutine method to survey the infiltration of leukemia. The infiltration of leukemic cells invarious organs was tracked by the expression of Rap1GAP and MMP-9genes withRT-PCR method.【Results】[Section1] The Rap1GAP expression in BMNCs was significantly lower in AML than innon-malignant blood diseases patients. In leukemic cell lines （HL-60, NB4, U937andSHI-1） Rap1GAP were also expressed very low, both in mRNA and protein level.Lentivirus vector pRRL-Venus-Rap1GAP was successfully constructed which can packagehigh titer virus particles with high transfection efficiency about80-90%. Two stableRap1GAP-transfected subclones of both HL-60and NB4cells proved by qRT-PCR and Western Blot were obtained through limited dilution method. qRT-PCR analysis showedthat the mRNA level of Rap1GAP increased about16.2,17.3,17.5and19.3folds in H1,H2, N1and N2cells, respectively. Western Blot proved that the level of Rap1GAPincreased significantly and GTP-Rap1was almost undetectable in theseRap1GAP-overexpressed cells. Up-regulated expression of Rap1GAP in HL-60and NB4cells does not influence the proliferation capability compared to empty control or widetype cells according to cell growth curve and colony forming assay. Neither the differenceof phospho-ERK or total ERK levels between Rap1GAP-transfected cells and theircounterparts was observed. During ATRA-induced differentiation of HL-60cells, theCD11b MFI of two Rap1GAP-transfected monoclones was1.4to2.1folds of thecounterpart cells each day （P<.05）. NBT assay also demonstrated the similar results.TPA-induction also resulted in much higher CD11b MFI in Rap1GAP-transfectedmonoclones at48h and72h （P<.05）, but the difference was not observed at24h. DuringATRA-induction of NB4cells, the CD11b MFI of two transfected monoclones was1.2to2.0folds of the counterpart cells at24h and48h, but the difference was not observed at72h.NBT assay was consistent with the CD11b results. After As₂O₃treatment, the cells stainedwith Annexin V-PE were much more in Rap1GAP-transfected cells than in the counterpart.The invasion rate of Rap1GAP-transfected HL-60cells was about5folds of the controlcells transfected with empty vector. Rap1GAP-transfected cells showed higher MMP-9secretion than the control cells, but MMP-2secretion difference was not observed. ThemRNA level of MMP-9was up-regulated in Rap1GAP-transfected HL-60cells （about12folds increase） compared with the corresponding control cells.[Section2] The mice of the control group manifested foodintake reducing, body weightlosing and emaciation in1-2weeks after infusion. Then the mice recovered gradually aftertwo weeks and survived more than60days. The mice of HL-60and MOCK groups alsoshowed the same manifestations during the first two weeks and recovered again, but6weeks later the manifestations appeared again. All the mice become prostration graduallyand died. The median survival time of all mice in HL-60group was （44.25±2.12）d and themedian survival time of MOCK group mice was （43.62±2.32）d. All the mice of H1and H2groups became sick earlier than the mice of HL-60and MOCK groups. The mice of H1and H2groups became sick again in4-5weeks after infusion, presenting foodintakereducing, body weight losing, emaciation and prostration gradually until dead. Three mice developed paralysis in both of the rear legs. The median survival time of mice in H1andH2group was （32.00±1.85）d and （33.37±2.50）d respectively. The difference of survivaltime in HL-60and MOCK groups mice was not observed. The survival time of R1and R2groups mice were shorter than the HL-60and MOCK group mice. The mice of R1and R2groups showed leukemic cells infiltration in meninges tissue and the genes of Rap1GAPand MMP-9were amplified by PCR method. The leukemic cells can be detected in thebone marrow smear of the paralyzed mice, but the infiltrations of leukemic cells in otherorgans were not obviously. The mice of HL-60and MOCK groups showed no apparentleukemic cells infiltrations in organs and Rap1GAP or MMP-9gene expression were notdetected.【Conclusion】（1） Decreased Rap1GAP expression was observed in BMNCs of AML patients as well asin leukemic cell lines including HL-60, NB4, U937and SHI-1cells.（2） Up-regulated expression of Rap1GAP do not affect myeloid leukemic cells growthability in vitro.（3） Up-regulated expression of Rap1GAP promotes the differentiation of NB4and HL-60cells induced by ATRA or TPA.（4） Up-regulated expression of Rap1GAP depresses survival ability of HL-60and NB4cells.（5） Up-regulated expression of Rap1GAP increased invasion ability of HL-60cells in vitroand in mouse model.In summary: Our results demonstrated that the expression of Rap1GAP wasdown-regulated in AML compared with the control group. Up-regulated expression ofRap1GAP （about17-19folds） in HL-60and NB4cells had no effect on cell growth ability,but promoted leukemic cells differentiation induced by ATRA or TPA. Increasedexpression of Rap1GAP also promoted cell apoptosis induced by As₂O₃. Transwell assayand leukemic mouse model showed that up-regulated expression of Rap1GAP increasedthe invasion ability of HL-60cells. These results initially studied the function of Rap1GAPin leukemia cell lines and showed that Rap1GAP may take part in the development ofAML. However, the exact mechanism of the effect of Rap1GAP in leukemogenesis needs further exploration.