[Inhibitory Effect of PKC412 Against Human Acute Leukemia Cell Line HL-60 Cells].

OBJECTIVE: To explore the effects and mechanisms of PKC412 inhibitor on proliferation and apoptosis of HL-60 cell line. METHODS: CCK-8 assay was used to detect the effect of PKC412 on the proliferation of HL-60 cells at different concentrations; Wright-Giemsa staining was used to estimated the effect of PKC412 on the apoptosis of HL-60 cells; the mRNA expression of BCL-2 and P53 genes was detected by qRT-PCR, the expression of BCL-2 and P53 proteins was detected by Western blot. HL-60 cells were injected into mouse caudal vein to construct acute myeloid leukemia model, PKC412 was administered to tail vein for 31.25 nmol/kg, normal saline was injected into the same site of the mice as control group, and the inhibitory effect of PKC412 on HL-60 cells in mice was observed. ELISA assay was used to detect the effect of PKC412 on the inflammatory factors of TNF-α and TGF-ß in tumor mice. RESULTS: PKC412 could inhibit the proliferation of HL-60 cell, which was in a dose dependent manner(r=0.9973) (IC50 was 0.31 µmol/L), and induce apoptosis of HL-60 cells. After HL-60 cell was treated by PKC412 for 48 h the expression of BCL-2 gene was down regulated(0.417±0.044 vs 0.933±0.033, t=9.347, P<0.001), the expression of P53 gene was up regulated(1.533±0.145 vs 1.050±0.161, t=2.231, P>0.05) as compared with control group. And the expression of BCL-2 protein was decreased, while the expression of P53 protein was increased. PKC412 could inhibited the growth of HL-60 tumor cells in vivo, the survival rate of mice after administration was 50% and the weight was increased as compared with that in control group(18.02±0.403 g vs 16.44±0.562 g, t=2.272, P=0.0356). The secretion of TNF-α and TGF-ß cytokine in serum and spleen cells in PKC412 group was significantly lower than that in control group (P<0.05). CONCLUSION: PKC412 can induce apoptosis of HL-60 cells by inhibiting the expression level of BCL-2 gene, PKC412 administration in vivo can inhibit the growth of the tumors.

Could cytokine release syndrome induce acute myelofibrosis in CD19 chimeric antigen receptor T cells therapy?

CAR-T cells therapy can give rise to most common and concerning two side effects - cytokine release syndrome (CRS) and neurotoxicity. But in our CD19 CAR-T cells therapy clinical trial, we observed 1 out of 17 patients with B-cell acute lymphoblastic leukemia (B-ALL) developed acute myelofibrosis(AMF) after grade IV CRS post to the CD19 CAR-T cells therapy. This finding suggests that the CAR-T cells therapy may have rare and serious AMF, which we should pay important attention to. Trial registration:NCT02968472. Registered 18 November 2016 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02968472.

[Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients].

OBJECTIVE: To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. METHODS: Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. RESULTS: ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). CONCLUSION: The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

Dys-psychological Stress Effect on Expressions of P53 and NFκBp65 in Human Ovarian Carcinoma In Vivo.

OBJECTIVE: To investigate the dys-psychological stress effect on the growth of subcutaneous xenotransplanted tumor in nude mice bearing human epithelium ovarian carcinoma, and the influence on P53 and NFκBp65 expressions. METHODS: The subcutaneous tumor xenografts were established by implanting human epithelium ovarian carcinoma tissues into nude mice and the dys-psychological stress model was established with restraint. The mice were randomized into the following four treatment groups with each group six mice respectively: tumor group (group A), normal saline intraperitoneal injection; tumor with stress group (group B), normal saline intraperitoneal injection; tumor therapy group (group C), cisplatin intraperitoneal injection; and tumor therapy with stress group (group D), cisplatin intraperitoneal injection. The expressions of P53 and NFκBp65 in tumor tissues were determined by Western blotting. RESULTS: The expressions of P53 and NFκBp65 in each restraint group were enhanced compared with the control groups (P<0.05). CONCLUSION: The dys-psychological stress may induce the high expressions of P53 and NFκBp65 proteins and further promote tumor growth.

[Association between XRCC1 gene polymorphisms and DNA damage of workers exposed to formaldehyde].

OBJECTIVE: To investigate the association between polymorphisms of DNA repair gene XRCC1 and DNA damage in peripheral blood lymphocytes among workers exposed to formaldehyde. METHODS: One hundred and fifty-one workers exposed to formaldehyde from plywood factories and one hundred and twelve workers without occupational exposure to formaldehyde were recruited into this study. DNA damage levels were measured by comet assay. The polymorphisms of XRCC1 gene were analyzed by polymerase chain reaction(PCR) with restriction fragment length polymorphism(RFLP) method. The multiple covariance analysis was used to compare olive trail moment and comet trail length adjusted confounding factors. RESULTS: In formaldehyde exposed workers, after ages, smoking and drinking status and occupational exposure level were adjusted, means of Olive trail moment and comet trail length in the subjects with variant genotype at Arg280 His site (geometric means 4.30 and 13.42 respectively) were higher than subjects with wild type homozygote (geometric means 3.38 and 11.71 respectively), the differences were significant (Olive trail moment: P < 0.05, comet trail length: P < 0.01) . No associations between the polymorphisms at other three sites in XRCC1 gene and means of olive trail moment and comet trail length in exposure workers were found. CONCLUSION: The polymorphisms of XRCC1 gene may modulate the effects of DNA damages induced by formaldehyde in workers.

[Early genetic effects on workers occupationally exposed to formaldehyde].

OBJECTIVE: To investigate the DNA and chromosome damage in peripheral blood lymphocyte of workers occupationally exposed to formaldehyde (FA). METHODS: All 151 workers occupationally exposed to FA from two plywood factories and 112 workers without occupational FA exposure working in a machine manufactory were recruited into this study. Comet assay and cytokinesis-block micronucleus technique was used to evaluate the DNA and chromosomal damage of peripheral blood lymphocyte. The air FA samples were collected with SKC 224-PCXR8 air samplers. Gas chromatography was used to analyze the FA level. Personal information including occupational history, age, sex, smoking and drinking status was collected by the questionnaire. RESULTS: The time weighted average concentration (TWA) of FA in the working environment of FA-exposed workers (range 0.10 - 7.88 mg/m(3)) was higher than those in controls (< 0.01 mg/m(3)). The olive tail moment (Olive TM) in low FA-exposed workers [3.03 (2.49 - 3.67)] was lower than that in high FA-exposed workers [3.95 (3.53 - 4.43)], but higher than that in controls [0.93 (0.78 - 1.10)], the differences were statistical significant (P < 0.05). Comet trail length in FA-exposed workers were significantly higher than that in controls [6.78 (6.05 - 7.60)], but no significant differences ware found between the high FA-exposed workers [12.59 (11.80 - 13.43)] and the low FA-exposed workers [11.25 (10.12 - 12.50)]. The frequency of micronuclei per 100 binucleated cells in low FA-exposed workers (0.41 +/- 0.25) was lower than that in high FA-exposed workers (0.65 +/- 0.36), but higher than that in controls (0.27 +/- 0.13), the differences were statistical significant (P < 0.05). The increased tendencies with the exposure levels were found in those three indices. In stratification analysis, the same results were found. CONCLUSION: In the current FA exposure levels, the DNA and chromosomal damage in peripheral blood lymphocyte might be induced by FA exposure, and be increased with the levels of exposure.