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RKIP loss aggravates RAS-driven leukemogenesis
comparisons in primary patient samples, we used the Wilcoxon- Mann-Whitney test for continuous variables and the Fisher’s Exact test for dichotomous variables. The effects of RKIP expression on survival were tested by the log-rank test. SPSS version 22.0 (SPSS Inc.) was employed for these calculations. All tests were two-sided and P<0.050 was considered statistically significant.
Study approval
The study was reviewed and approved by the institutional review board (28-481 ex 15/16) and conducted in accordance with the Declaration of Helsinki. Mouse experiments were approved by the Federal Ministry for Science, Research and Economy (GZ: BMWF-66.010/0050-II/3b/2013).
Results
RAF kinase inhibitor protein expression is high in hematopoietic stem and progenitor cells and lymphoid cells but low in cells belonging to the myeloid lineage
Hypothesizing that RKIP loss is a driver for
myelomonocytic lineage commitment, one would assume that cells with myelomonocytic differentiation would demonstrate lower RKIP expression levels. In a first step, we therefore employed an HL-60 in vitro differentiation model. HL-60 is an undifferentiated AML cell line and can be forced into the myelomonocytic lineage by addition of 1,25 dihydroxyvitamin D3.35 Interestingly, we observed that 1,25D3-induced differentiation in these cells was accompanied by a significant decrease in the amount of RKIP protein (P=0.002) (Figure 1A). We then aimed to delineate RKIP expression in human hematopoiesis by studying CD34+ HSPC derived from three umbilical cord blood specimens on the one hand, as well as mature lym- phocytes, granulocytes and monocytes from four healthy individuals on the other. Due to the restricted cell num- bers available, we studied RKIP mRNA by the means of quantitative-polymerase chain reaction (qPCR) in these experiments. However, we have previously shown that decreased expression of RKIP at the protein level is accom- panied by its downregulation at the mRNA level as well.12,20-22 In accordance with HL-60 data, RKIP was
A
BC
Figure 1. RAF kinase inhibitor protein (RKIP) expression is low in cells belonging to the myeloid lineage. (A) Immature HL-60 acute myeloid leukemia (AML) cells were treated with 100nM 1,25-dihydroxyvitamin D3 (1,25D3) for 48 hours to induce myeloid differentiation. Immunoblot analysis demonstrates a decrease in RKIP protein expression in 1,25D3 treated cells. The graph represents the mean of three independent experiments ±Standard Deviation; expression values are given as x-fold expression of the HL-60 control. Statistical significance was evaluated using Student’s t-test. (B) Box plots showing RKIP mRNA expression, studied via quan- titative polymerase chain reaction in CD34+ hematopoietic stem and progenitor cells (HSPC) (CD34+, n=3), lymphocytes (B-lymph, n=4), monocytes (Mono, n=4) and granulocytes (Granulo, n=4) from healthy donors. In comparison to HSPC, RKIP mRNA expression is significantly reduced in monocytes and granulocytes, while no significant difference was observed for lymphocytes. Graphs denote the x-fold RKIP expression levels of NB4 cells, which were used as a calibrator and arbitrarily set to a value of 1. P-values were calculated using Student’s t-test. (C) Box plots showing that the expression of RKIP mRNA in differentiated cells belonging to the myeloid lineage (MMC) is also decreased in mice. These data were generated by re-analysis of a previously published murine microarray gene expression profiling dataset.31 HSPC included long-term hematopoietic stem cells (lin–, Sca+, kit+, CD34–), short-term hematopoietic stem cells (lin–, Sca+, kit+, CD34+), LSK (lin–, Sca+, kit+) and hematopoietic progenitor cells (lin–). Myelomonocytic cells (MMC) included Gr-1+ neutrophils and Mac-1+ monocytes/macrophages. Statistical significance was cal- culated by Student’s t-test.
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