Hemopoietic Progenitor Cells from the Bone Marrow of Serpinb3A-/- Mice Are Radioresistant

Introduction: Serpins are a superfamily of serine proteases that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit multiple peptidases. Currently 36 human serpins have been identified, but the roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). A majority of serpins inhibit serine proteases but serpins that inhibit lysosomal cytosine peptidases, granzymes, serine protease and calpains. Previous studies have shown that serpins play a critical role in cell survival by blocking lysosomal induced necrosis injury. Serpinb3A (Antichymotrypsin) has been identified as an inhibitor of the serine peptidases, chymotrypsin and cathepsin G. We now report that hematopoietic progenitor cells in the bone marrow of Serpinb3A-/- mice are radioresistant.

Materials & Methods: Serpinb3A-/- and control Balb/c mice were obtained from Charles River and were housed five per cage according to University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) regulations. All protocols were approved by the University of Pittsburgh IACUC. Veterinary care was provided by the Division of Laboratory Animal Research of the University of Pittsburgh. Whole bone marrow was obtained by flushing the marrow from the femurs of sacrificed Serpinb3A-/- and Balb/c mice. Single cell suspensions were irradiated to doses ranging from 0 to 8 Gy using a at 70 cGy/min using a Shepherd Mark 1 137Cs γ-ray source (J.L. Shepherd, San Fernando, CA, USA). The cells were plated in 0.8% methylcellulose containing hematopoietic growth supportive medium (StemCell Technology product #3134) at varying densities in quadruplicate in Linbro plates (Fisher Scientific, Pittsburgh, PA, USA) and incubated at 37°C and 5% CO2 for 9-11 days at which time the number of colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 5 experiments was analyzed using linear quadratic and single-hit, multi-target models.

Results: CFU-GM from Serpinb3A-/- mouse marrow showed an increase in radiation resistance with an increase in the shoulder of the irradiation survival curve compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). There was no significant difference in the Do for CFU-GM of the two mouse strains (Do = 1.48 ± 0.19 Gy for Serapinb3A-/- mice and 1.56 ± 0.06 Gy for Balb/c mice, p = 0.0997.

Conclusions: The data support a possible role for the loss of Serpinb3a in providing a cell survival advantage for hematopoietic progenitor cells exposed to lethal doses of ionizing irradiation. This would suggest that the Serpinb3a target protease helps repair the cellular injury induced by ionizing radiation. Alternatively, the germ line loss of Serpinb3a may up-regulate the expression of other clade B serpins known to protect cells from injury. Ongoing studies will help determine the nature of this protective effect against radiation-induced cell death.