Abstract 1306

Hydroxyurea (HU) is a drug that is effectively used in the management of the β-hemoglobinopathies sickle cell disease and β-thalassemia by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear, as does the reason why some proportion of the patients failed to show a beneficial therapeutic response. A better understanding of how hydroxyurea acts at the molecule levels and induces fetal hemoglobin production would offer targeted molecular therapy with higher specificity, and less toxicity and may provide alternative effective therapies to current non-responders. Secretion-associated and ras-related protein (SAR1) is a small guanosine triphosphate (GTP)-binding protein that is induced by HU and closely mimics the known effects of HU on erythroid cells, including gamma-globin induction and cell-cycle regulation. Here we report of our further investigation on whether SAR1 is an obligatory mediator to the HbF-inducing effect of HU and other therapeutic cytotoxic agents. Our results indicate that blocking SAR1 expression not only significantly lowered the basal and HU-elicited HbF production in CD34+ and K562 cells (see table), it also abolished HU-mediated S-phase cell-cycle arrest (see table) and cell apoptosis (P<0.001).Transcriptional factor BCL11A, which controls globin switching and suppresses HbF, is involved in SAR1 and HU regulated gamma-globin expression, and SAR1 is required for HU, 5-AZA and Ara-C mediated BCL11A reduction. We also found HU acts through the interaction of NFκB with its conjugated binding site on SAR1 promoter to regulate its transcriptional expression. Our data show that SAR1 is not only sufficient, but indeed necessary and central, to HU's beneficial effects. Our data suggests that SAR1 is not only sufficient, but indeed necessary and central, to HU's erythroid effects. Further exploration of the mechanistic details underlying SAR1 effects may reveal novel therapeutic targets for the beta-globin disorders.

Mock
Control shRNA or microRNA
SAR1 shRNA or microRNA
HU−HU+HU−HU+HU−†pHU+‡p
HbF positive CD34 + cells   27.00 ± 1.01% 50.70 ± 1.88% 20.59 ± 0.76% 0.003 31.77 ± 1.17% 0.00001 
HbF positive K562 cells 18.21 ± 0.21% 41.00 ± 1.41% 19.72 ± 2.11% 43.37 ± 0.2% 14.29 ± 1.2% 0.004 26.63 ± 1.23% 0.00002 
Fraction of S-phase cells 24.94 ± 0.84% 32.38 ± 0.59% 24.08 ± 0.59% 34.26 ± 0.54% 24.88 ± 0.4% 0.097 27.64 ± 0.25% 0.00192 
Mock
Control shRNA or microRNA
SAR1 shRNA or microRNA
HU−HU+HU−HU+HU−†pHU+‡p
HbF positive CD34 + cells   27.00 ± 1.01% 50.70 ± 1.88% 20.59 ± 0.76% 0.003 31.77 ± 1.17% 0.00001 
HbF positive K562 cells 18.21 ± 0.21% 41.00 ± 1.41% 19.72 ± 2.11% 43.37 ± 0.2% 14.29 ± 1.2% 0.004 26.63 ± 1.23% 0.00002 
Fraction of S-phase cells 24.94 ± 0.84% 32.38 ± 0.59% 24.08 ± 0.59% 34.26 ± 0.54% 24.88 ± 0.4% 0.097 27.64 ± 0.25% 0.00192 
†

p compared with control HU-

‡

p compared with control HU+

View Large

Disclosures:

No relevant conflicts of interest to declare.

Topics:
cytotoxin, fetal hemoglobin, gamma-globins, hydroxyurea, micrornas, azacitidine, beta-globin, cd34 antigens, cytarabine, globins

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2011 by The American Society of Hematology
2011
Sign in via your Institution