Abstract

Abstract 2360

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of granulocyte precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×106 cells/ml in LCL-C vs 5×105 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi.

Conclusion:

Overexpressed Gal1 was found from SDS patient's derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.