The Regulation Of Shh Signaling In Chronic Myeloid Leukemia Stem/Progenitor Cells

【Purpose】This study aimed to explore the activity of Shh signaling in chronic myeloid leukemia by analysis of the key components of Shh signaling in chronic myeloid leukemia and healthy controls. And to further find the correlation of Shh signaling with CML disease progression by analysis of the activity of Shh signaling in CML-CP, CML-AP, CML-BP cases.[Methods] CML-CP, CML-AP, CML-BP cases and healthy controls were collected, bone marrow mononuclear cells were isolated. The key components of Shh signaling SHH, SMO, PTCH1, GLI1 in chronic myeloid leukemia cases and healthy controls were assayed by RT-PCR and more sensitive Real time PCR (RQ-PCR) analysis as well.【Results】Differentiated expression of Shh signaling components was observed between cases, especially in chronic phase. The positive regulators of Shh signaling were up-regulated (more than 2 times) in 50% CML-CP samples,70% CML-AP and 80% CML-BP samples, which meant the presence of an active Shh pathway in chronic myeloid leukemia. More samples in CML-AP/BP showed up-regulation of SHH, SMO, GLI1 than in CML-CP, which revealed that Shh signaling was correlated with chronic myeloid leukemia disease progression.【Conclusion】Shh signaling was activated in chronic myeloid leukemia, and correlated with CML disease progression. 【Purpose】This study aimed to explore the activation status of Shh signaling in CML stem /progenitor cells.【Methods】CD34 positive rates in CML-CP, CML-AP and CML-BP samples were measured by FACS. Co-expression of CD34/c-kit and Shh protein was detected in CML-AP, CML-BP bone marrow smears by immunostaining. CD34+cells were purified by MACS. The efficiency of MACS was measured by FACS. mRNA expression of SHH, SMO, PTCH1, GLI1 in both CD34+and CD34- cells was assayed by Real time PCR, and the expression of the four components in K562 cells was analyzed by RT-PCR. The expression of Shh protein was detected in CD34+cells, CD34- cells, CML bone marrow stromal cells and K562 cells by immunostaining.【Results】CD34 positive rates in CML-CP, CML-AP and CML-BP samples were in the range of 0.2%～2.0%,4.0%～10.0%,12.5%-97.5%, respectively. Co-expression of CD34 /c-kti and Shh protein was observed in bone marrow smears. Shh protein was over-expressed in cells with high CD34/c-kit expression, and vice versa. The efficiency of MACS was＞95%. CD34+cells showed higher levels of SHH, SMO, GLI1 mRNA than CD34- cells. Co-expression of CD34 and Shh protein was also observed in purified CD34+ and CD34" cells. CML bone marrow stromal cells showed low levels of Shh protein. SHH, SMO, PTCH1, GLI1 were all expressed in K562 cells, and differentiate expression of Shh protein was observed in K562 cells:Some cells showed high levels of Shh protein, while the others showed low levels of Shh protein.【Conclusion】There was an active Shh signaling in CML stem/progenitor cells. Shh protein in autocrine accounted for auto-activated Shh signaling in CML stem/progenitor cells. 【Purpose】This study aimed to explore the effect of Shh signaling on the survival and proliferation of CML stem/progenitor cells.【Methods】CD34+ cells were isolated from bone marrow samples of CML-AP, CML-BP cases by MACS. Both CD34+ and CD34- cells were cultured in the medium with lOng/ml IL-3,10ng/ml IL-6,50ng/ml SCF. Exogenous Shh peptide and the inhibitor of Shh signaling, cyclopamine, were respectively administered to CD34+ and CD34- cells. Cell proliferation was detected by MTT assay. K562 cells were divided into four groups(1) normal controls (2) Shh group (500ng/ml) (3) cyclopamine group (10μM) (4) Shh (500ng/ml)+cyclopamine(10μM) goup, cell proliferation was detected by MTT assay after 24h,48h. Different doses of 5E1 monoclonal antibody and cyclopamine were administered to K562 cells respectively. Cell number was observed by light microscope after 24h. Cell apoptosis rate was detected by Annexin V/PI, and cell cycle was assayed by PI method. The small interference RNA (siRNA) was synthesized in vitro. K562 cells were transfected with GLI1 siRNA by the way of lipofection (lipofectamine 2000). Non-specific siRNA transfected cells were used as controls. Transfection efficiencies of siRNA were detected by flow cytometry and the best siRNA concentration was detected. The silencing effect of siRNA was demonstrated by Real time PCR and western blot analysis. Cell proliferation was measured by MTT method after 24h,48h.【Results】After 72h of exogenous Shh peptide administration, CD34- cells expanded 1.18±0.04 fold, while CD34+ cells expanded 1.64±0.11 fold, much higher than CD34- cells (p<0.01). After 72h of cyclopamine administration, the number of CD34- cells was 0.75±0.03 fold of original cell number, while the number of CD34+ cells was 0.88±0.04 fold, much lower than CD34- cells. After 24h, the cell number of K562 cells in control group was 1.57±0.09 fold of the seeded cell number, it was1.63±0.10 fold in Shh group (P=0.13, compared to controls),1.05±0.08 fold in cyclopamine group (P＜0.01), and 1.17±0.09 fold in Shh+cyclopamine group (P＜0.01). After 48h, the cell number of K562 cells in control group was 1.32±0.08 fold of the seeded cell number, it was 1.78±0.07 fold in Shh group (P＜0.01, compared to controls),0.64±0.07 fold in cyclopamine group (P< 0.01), and 0.72±0.10 fold in Shh+cyclopamine group (P<0.01). Exogenous Shh peptide induced proliferation of K562 cells and cyclopamine induced cell apoptosis in a time-dependent way. Both 5E1 antibody and cyclopmine induced K562 cell apoptosis and G2/M cell cycle arrest in a dose-dependent way. The mRNA level of GLI1 in GLI1 siRNA group was (52.60±3.57)%(P＜0.01), (88.83±5.21)%(P=0.01),1.36±0.11 fold (P＜0.01) of control after 24h,48h,72h, respectively. The protein level of Glil in GLI1 siRNA group was (88.60±6.13)%(P=0.02), (79.31±5.58)%(P＜0.01) of control after 24h,48h respectively. Total cell number in GLI1 siRNA group was (94.41±3.58)%(P=0.05) of control after 24h, and (90.22±3.34)%(P＜0.01) of control after 48h, which demonstrated that GLI1 siRNA inhibited the expression of GLI1, and induced e cell apoptosis.【Conclusion】Auto-activated Shh signaling maintained survival and proliferation of CML stem/progenitor cells. Inhibition of Shh signaling induced apoptosis and cell cycle arrest of CML stem/progenitor cells. [Purpose] This study aimed to explore the mechanism of Shh signaling in the regulation of CML stem/progenitor cells by analysis of the relationship and interaction of Shh signaling with Wnt signaling.【Methods】CD34+and CD34" cells were isolated from CML-AP and CML-BP bone marrow samples by MACS. Bone marrow stomal cells were cultured. Co-expression of Shh andβ-catenin protein in CD34+cells, CD34- cells, bone marrow stromal cells and K562 cells were detected by two-colour immostaining. The key components of both Shh signaling and Wnt signaling were detected in K562 cells by RT-PCR analysis. K562 cells were treated as follows:(1) normal control group (2) Shh antibody group (10ug/ml) (3) Shh antibody (10μg/ml)+Wnt3a (500ng/ml) group (4) Wnt3a group (10μg/ml) (5) Wnt3a antibody (10μg/ml)+Shh-N (500ng/ml) group. Annexin V/PI method was performed to detect the apoptosis rate of each group. The mRNA expression of GLI1, P-CATENIN, C-MYC in K562 cells treated with l0μg/ml Shh antibody for 24h were detected by RT-PCR. More sensensitive real time PCR was used to detect the mRNA level of GLI1,β-CATENIN, C-MYC, P21,BCL-2 in K562 cells treated with10μg/ml Shh antibody for 24h.【Results】Co-expression of Shh and P-catenin protein was observed in CD34+cells, CD34- cells, bone marrow stromal cells and K562 cells. The key components of Shh signaling and Wnt signaling were expressed in K562 cells. The apoptosis rate of K562 cells in normal control group after 24h was 4.15±1.02%, and it was15.88±2.87% in Shh antibody (10μg/ml) group,8.41±1.38%in Shh antibody (10μg/ml)+Wnt3a (500ng/ml) group (p＜0.01, compared to Shh antibody group),17.34±3.15% in Wnt3a antibody (10μg/ml) group,8.41±1.38% in Wnt3a antibody (10μg/ml)+Shh-N (500ng/ml) group (P=0.60, compared to Wnt3a antibody group). Wnt3a could salvage the apoptosis of K562 cells induced by Shh antibody, while Shh-N could not revert the apoptosis of K562 cells induced by Wnt3a antibody, which meant that Wnt signaling acted downstream of Shh signaling. The mRNA level of GLI1,P-CATENIN,C-MYC,BCL-2 decreased 7.92±0.89, 0.21±0.14,4.95±0.57 and 2.55±0.21 fold, respectively, and the mRNA level of P21 increased 11.57±1.23 fold in K562 cells treated withlOμg/ml Shh for 24 h, which showed that the expression of GLI1, C-MYC, BCL-2 was downregulated, and the expression of P21 was upregulated, but there was no obvious change in the expression ofβ-CATENIN.【Conclusion】Wnt signaling acted downstream of Shh signaling, and Shh signaling maintained survival and proliferation of CML progenitor cells through action on the target genes of Wnt signaling.