Louis, MO, USA) (1 mol/L) for 30 minutes at 37C


Louis, MO, USA) (1 mol/L) for 30 minutes at 37C. were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. 0.001) (Figure ?(Figure1A)1A) in chemotherapy resistant cell lines (K562/ADM and HL60/ADM). Among the primary cells, Cells from patient 2 had the lowest expression of endogenous pig7 while those from patient 4 had the highest expression (* 0.001) (Figure ?(Figure1B).1B). After transfection with lentivirus Plenti6.3-PIG7, the mRNA and protein expressions of pig7 were both significantly increased, reaching very high levels in all cells. However, protein expression of pig7 showed no significant differences in either the four kinds of cell lines or in the five cases of primary cells. Thymopentin Overexpression of pig7 disproportionately enhanced the chemosensitivity of these cells, with the exception of the HL60 cell line. Among the four cell lines, the IC50 values of VP16 and ADM at 48 h for K562/ADM cells, which had the lowest expression of endogenous pig7, were reduced from 407.3 g/ml and 4.01 g/ml for the Plent6.3 group to 79.6 g/ml and 0.28 g/ml for the Pig7 groups, respectively. Their chemosensitivity also increased 5.1- and 14.3-fold, respectively. HL60 cells had a relatively high endogenous expression of pig7 and the 48 h IC50 values of both VP16 and ADM were not significantly changed (** 0.05) (Figure ?(Figure2A).2A). In the five cases of primary cells, patient 2 had the lowest expression of endogenous pig7 and also had decreased IC50 at 48 h for both VP16 and ADM (from 29.3 g/ml and 1.19 g/ml to Thymopentin 6.7 g/ml and 0.12 g/ml, respectively). Their chemosensitivity increased 4.3- and 9.9-fold, respectively. In contrast to patient 2, patient 4 had the highest expression Thymopentin of endogenous pig7 and did not have significant changes in IC50 of either VP16 or ADM at 48 h. Their chemosensitivity only increased 1.3- and 1.6-fold, respectively (Figure ?(Figure2B).2B). Annexin V staining assay indicated that the largest increase in the apoptosis rate (Annexin V+/7-AAD+ cells%) occurred in K562/ADM and patient 2 primary cells treated with both Plent6.3-PIG7 and VP16 (39.7 4.7% VS 16.9 3.9%, 50.2 4.8% VS 25.4 3.1%, respectively, * 0.01) (Figure ?(Figure3A).3A). The necroptosis rate increase (Annexin V?/7-AAD+ cells%) of these cells was also the highest (17.9 2.3% VS 5.9 0.7%, 22.7 3.7% VS 7.6 1.3%, respectively, * 0.01) (Figure ?(Figure3B).3B). However, in HL60 and patient 4 primary cells, the apoptosis rate was not significantly changed (24.2 3.4% VS 22.7 3.1%, 31.2 3.3% VS 29.8 4.1%, respectively, ** 0.05) (Figure ?(Figure3A).3A). The increase in the necroptosis rate in these cells was also very mild (10.2 1.7% VS 7.9 1.3%, 9.1 1.5% VS Thymopentin 7.4 1.7%, respectively, ** 0.05) (Figure ?(Figure3B).3B). Collectively, these results indicate that the chemosensitivity promoting effect of pig7 is widely varied in both different leukemia cell lines and primary cells. Moreover, the expression level of endogenous pig7 may have a strong negative correlation with this observed chemosensitive effect. Open in a separate window Figure 1 Expression of pig7 mediated by lentivirus infection(A) Endogenous expression of pig7 in K562/ADM and HL60/ADM cell lines was significantly lower than in K562 and HL60 cell lines (* 0.01). (B) Patient 2 had the lowest expression of endogenous pig7 and Patient 4 had the highest expression (* 0.001). In all cells, high levels of pig7 product could be detected in the plent6.3-PIG7 group by RT-PCR and Western blot at 48 h post-lentiviral infection. There was no significant difference ACTN1 in pig7 protein expression ( 0.05). Open in a separate window Figure 2 MTT assay and decreased IC50 in cells infected for 48 h with Plent6.3-PIG7 in combination with.