Survivin overexpression alone does not alter megakaryocyte ploidy nor interfere with erythroid/megakaryocytic lineage development in transgenic mice

Abstract

IntroductionThe megakaryocyte (MK) is a unique hematopoietic cell that undergoes multiple rounds of polyploidization in which the cellular DNA content can reach up to 128N, with 2N being normal diploid content. The process of polyploidization in MKs is thought to play an important role in supporting the large increase in size of mature MKs, which are capable of producing thousands of platelets upon fragmentation, 1 however, much of this process remains enigmatic.Recent studies have implicated a member of the chromosome passenger complex, survivin, as being mislocalized or downregulated at the protein or mRNA level during mitosis of murine MKs of high ploidy. 2,3 In contrast, protein levels and localization of survivin was reported to be typical during mitosis of human megakaryocytes (which, in culture, are typically of low ploidy). 4 Retroviral overexpression of survivin in vitro decreased murine MK ploidy level and number of MK colonies in MK colony assays in comparison to untreated MKs. 3 Similarly, primary vascular smooth muscle cells, which naturally undergo polyploidization in culture, were also shown to decrease their ploidy level upon survivin overexpression. 5 Lately, an increasing number of studies have identified survivin expression in normal adult cells. 3,6,7 Leung et al 7 recently reported on the requirement of survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells. While survivin expression has been demonstrated to be essential in various hematopoietic cell types, the effect of its ectopic expression has not yet been analyzed in vivo. We report the establishment of 2 tissue-specific hematopoietic survivin transgenic models, one overexpressing survivin in both the erythroid and megakaryocyte lineages, and the other solely in the megakaryocytic lineage. MethodsTransgenic mouse production, MK purification, flow cytometry, and MK colony assay Detailed procedures are outlined in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Reverse transcriptase-polymerase chain reaction of transgenic mRNATotal RNA was harvested from mouse bone marrow cells using TRIzol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Transgenic mRNA in total bone marrow was detected via reversetranscriptase polymerase chain reaction (RT-PCR) as previously described. 8 ␤-galactosidase assayVisualization of ␤-galactosidase transgene activity in MKs was performed as previously described 8 using freshly isolated bone marrow (BM) from wild-type and PF4-surv mice. Western blot analysisWestern blotting was performed as previously described. 8 Primary antibodies used were anti-survivin, anti-CD41, anti-HSC70 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-10811, sc-15328, and sc-7298, respectively), and anti-␤-actin (Sigma-Aldrich, St Louis, MO; A5441). Acetylcholinesterase assayMK number was calculated based on a positive signal resulting from MK-specific acetylcholinesterase activity...

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

“…p21 −/− [41] and survivin-deficient [45] or -over-expressing [46] mice have normal steady state Mk development and platelet levels. Nevertheless, loss of p21 has been linked to enhanced polyploidization in vitro [47].…”

A new role for the A2b adenosine receptor in regulating platelet function

“…A2bAR mRNA expression was measured by quantitative reverse‐transcription polymerase chain reaction. In vitro study (upper panel): mRNA was prepared from freshly isolated MKs or MKs cultured for 3 days , derived from wild‐type (WT) mice as in [16], purified with a CD41‐based column as described in . In vivo study (lower panel): after treatment with TPO or phosphate‐buffered saline (0.05 μg g −1 , tail vein injection) for 4 days, the MKs were isolated and purified as above.…”

“…(B) β‐Galactosidase (β‐Gal) staining of MKs derived from WT or A2bAR knockout (KO) mice and viewed at × 600 magnification with an Olympus IX70 microscope combined with a Hamamatsu charge‐coupled device camera (C4742‐95). MKs were isolated and cultured as in [16], and then subjected to β‐Gal staining (see ). Blue staining in A2bAR KO MKs (here displayed as a darken shade) indicates the expression of β‐Gal gene driven by the A2bAR gene promoter (left panel).…”

“…Bone marrow cells were isolated and cultured as described previously [16]. MKs were purified by the MACS magnetic bead purification system (Miltenyi Biotech, Bergisch Gladbach, Germany), as described in [16] and Doc. S1.…”

Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A

“…These results are similar to those of previous studies, 40 , 46 but we used IDLVs, a safer system, to achieve a safer cancer therapy. However, since Survivin protein overexpression was also detected in megakaryocyte and erythrocytes, and it is also up-regulated by β-catenin, 60 which is activated during erythropoiesis process. 61 Therefore, a long-term observation for side effect on hematopoiesis and large-scale experiments using our Survivin promoter-driven IDLV system should be performed to validate the safety of our system before medical trials.…”