A single copy of large tumor suppressor 1 or large tumor suppressor 2 is sufficient for normal hematopoiesis.

BACKGROUND: Hematopoietic stem cells (HSCs) have the ability to differentiate into all subsets of blood cells and self-renew. Large tumor suppressor 1 (LATS1) and large tumor suppressor 2 (LATS2) kinases are essential for cell cycle regulation, organism fitness, genome integrity, and cancer prevention. Here, we investigated whether Lats1 and Lats2 are critical for the maintenance of the self-renewal and quiescence capacities of HSCs in mice. METHODS: Quantitative reverse transcription-polymerase chain reaction was used to determine the expression levels of Lats1 and Lats2 in subsets of progenitor cells and mature bone marrow cells. A clustered regularly interspaced short palindromic repeats system was used to generate Lats1 or Lats2 knockout mice. Complete blood cell counts were used to compare the absolute number of white blood cells, lymphocytes, monocytes, neutrophils, and platelets between Lats1 or Lats2 heterozygotes and littermates. Flow cytometry was used to assess the size of hematopoietic progenitor cells (HPCs) and HSC pools in Lats1 or Lats2 heterozygotes and littermates. The comparison between the two groups was analyzed using Student's t test. RESULTS: Lats1 and Lats2 were widely expressed in hematopoietic cells with higher expression levels in primitive hematopoietic cells than in mature cells. Lats1 or Lats2 knockout mice were generated, with the homozygotes showing embryonic lethality. The size of the HPC and HSC pools in Lats1 (HPC: wild-type [WT] vs. heterozygote, 220,426.77 ±â€Š54,384.796 vs. 221,149.4 ±â€Š42,688.29, P = 0.988; HSC: WT vs. heterozygote, 2498.932 ±â€Š347.856 vs. 3249.763 ±â€Š370.412, P = 0.105) or Lats2 (HPC: WT vs. heterozygote, 425,540.52 ±â€Š99,721.86 vs. 467,127.8 ±â€Š89,574.48, P = 0.527; HSC: WT vs. heterozygote, 4760.545 ±â€Š1518.01 vs. 5327.437 ±â€Š873.297, P = 0.502) heterozygotes were not impaired. Moreover, the depletion of Lats1 or Lats2 did not affect the overall survival of the heterozygotes (Lats1: P = 0.654; Lats2: P = 0.152). CONCLUSION: These results indicate that a single allele of Lats1 or Lats2 may be sufficient for normal hematopoiesis.

Zinc finger and BTB domain-containing protein 46 is essential for survival and proliferation of acute myeloid leukemia cell line but dispensable for normal hematopoiesis.

BACKGROUND: Zinc finger and BTB domain-containing protein 46 (Zbtb46) is a transcription factor identified in classical dendritic cells, and maintains dendritic cell quiescence in a steady state. Zbtb46 has been reported to be a negative indicator of acute myeloid leukemia (AML). We found that Zbtb46 was expressed at a relatively higher level in hematopoietic stem and progenitor cells (HSPCs) compared to mature cells, and higher in AML cells compared to normal bone marrow (BM) cells. However, the role of Zbtb46 in HSPCs and AML cells remains unclear. Therefore, we sought to elucidate the effect of Zbtb46 in normal hematopoiesis and AML cells. METHODS: We generated Zbtb46 and Zbtb46Mx1-Cre mice. The deletion of Zbtb46 in Zbtb46Mx1-Cre mice was induced by intraperitoneal injection of double-stranded poly (I). poly (C) (poly(I:C)), and referred as Zbtb46 cKO. After confirming the deletion of Zbtb46, the frequency and numbers of HSPCs and mature blood cells were analyzed by flow cytometry. Serial intraperitoneal injection of 5-fluorouracil was administrated to determine the repopulation ability of HSCs from Zbtb46 and Zbtb46 cKO mice. The correlation between Zbtb46 expression and prognosis was analyzed using the data from the Cancer Genome Atlas. To investigate the role of Zbtb46 in AML cells, we knocked down the expression of Zbtb46 in THP-1 cells using lentiviral vectors expressing small hairpin RNAs targeting Zbtb46. Cell proliferation rate was determined by cell count assay. Cell apoptosis and bromodeoxyuridine incorporation were determined by flow cytometry. RESULTS: The percentages and absolute numbers of HSPCs and mature blood cells were comparable in Zbtb46 cKO mice and its Zbtb46 littermates (Zbtb46vs. Zbtb46 cKO, HPC: 801,310 ±â€Š84,282 vs. 907,202 ±â€Š97,403, t = 0.82, P = 0.46; LSK: 86,895 ±â€Š7802 vs. 102,210 ±â€Š5025, t = 1.65, P = 0.17; HSC: 19,753 ±â€Š3116 vs. 17,608 ±â€Š3508, t = 0.46, P = 0.67). The repopulation ability of HSCs from Zbtb46Mx1-Cre mice was similar to those from Zbtb46 control (P = 0.26). Zbtb46 had elevated expression in AML cells compared to total BM cells from normal control. Knockdown of Zbtb46 in THP-1 cells led to a significant increase in cell apoptosis and reduced cell growth and proliferation. CONCLUSION: Collectively, our data indicate that Zbtb46 is essential for survival and proliferation of AML cells, but dispensable for normal hematopoiesis.

[Effect of Apollon siRNA combined with tetramethylpyrazine on proliferation and apoptosis of leukemia K562 cells].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA) silencing Apollon gene combined with tetramethylpyrazine (TMP) on the proliferation and apoptosis of human chronic myeloid leukemia cell line K562. METHODS: K562 cells were divided into blank control, negative control, and RNA interference (RNAi) group. For the RNAi group, the pGPHI-GFP-Neo-Apollon eukaryotic expression vector based on the best Apollon siRNA fragments screened out in previous experiments was constructed; the blank control group received no treatment, and the negative control group was transfected with negative plasmid vector. The mRNA and protein expression of Apollon was measured by RT-PCR and cell immunofluorescence, respectively. Additionally, TMP (320 µg/mL) was applied to set TMP, TMP+negative control, and TMP+RNAi groups. The cell viability and apoptosis rate were determined by MTT assay and flow cytometry, respectively. RESULTS: The constructed vector was stably expressed in K562 cells. The RNAi group had significantly lower mRNA and protein expression of Apollon than the blank control group and negative control (P<0.05). The RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the blank contorl group (P<0.05). The TMP+RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the RNAi, and TMP groups (P<0.05). CONCLUSIONS: Apollon siRNA can significantly inhibit the proliferation and promote the apoptosis of K562 cells, and the addition of TMP can further increase the proliferation inhibition rate and apoptosis rate, suggesting that siRNA technology combined with drugs has a significant potential value in the treatment of leukemia.