Deregulation of vital mitotic kinase–phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia

Abstract

Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

Aplastic anemia, cellular and molecular aspects

“…Phospho Chk‐1 (Ser317), plays an important role in initiating proliferation and re‐entering the cell division process. Decreased regulation of phospho Chk‐1 (Ser317) in response to BU and CP toxicity in the aplastic BM may be responsible for cellular inefficiency in overcoming stress (Chatterjee et al, 2016).…”

“…DNA damage response (DDR) affects various cellular signaling pathways including protein kinase‐B (PKB)/GSK‐3ß pathway and can lead to abnormal cell cycle regulation and apoptosis. Apoptosis is activated by activation of caspase‐3 in addition to GSK‐3ß in aplastic HSPC (Chatterjee et al, 2016). In peripheral T cells of untreated AA patients, Notch‐1 IC (the active form of Notch‐1) is significantly elevated and binds to the TBX21 promoter, which indicates that Notch‐1 directly regulates the gene encoding T‐BET.…”

Quercetin induces autophagy in myelodysplastic bone marrow including hematopoietic stem/progenitor compartment

“…Finally, the cells were washed in PBS and analyzed in BD FACS Calibur flowcytometer (Becton Dickenson) using Cell Quest Pro software (v9.1 Becton Dickinson). The mean fluorescence intensity (MFI) data (10 000 events) obtained flowcytometrically was analyzed by virtual gating in the low FSC and low SSC zone as previously claimed to be hematopoietic stem/progenitor (HSPC) cell rich zone 52‐56 …”

“…The antioxidant status was determined by checking the level of expression of anti SDF1 and anti SOD2 antibodies in the quercetin treated and untreated bone marrow cells. The data was analyzed in the same low FSC/low SSC zone as described earlier 52‐56 …”

Aberrant Wnt Signaling Pathway in the Hematopoietic Stem/Progenitor Compartment in Experimental Leukemic Animal

“…Flow cytometry was performed to analyze expression profile of Wnt signaling components in control and leukemic condition as per our previously described protocol (Chattopadhyay et al 2016a;Daw et al 2016;Chatterjee et al 2016c;Chattopadhyay et al 2016b). 2X10 6 PFA fixed control and leukemic BM cells were distributed in polystyrene tubes and stained with following primary antibodies: anti-Wnt5a and anti-Wnt3a Cell membrane permeabilization was performed by 90% chilled methanol prior to antibody staining for detection of intracellular signaling molecules (except for surface proteins such as Fzd7, Fzd5, ROR2 and N-Cadherin).…”