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Date of Award

2004

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

James Blankenship

First Committee Member

Patrick Jones

Second Committee Member

John Livesey

Third Committee Member

Timothy Smith

Fourth Committee Member

David Fires

Abstract

This investigation focused on four aspects of hexamethylene bisacetamide's ( HMBA ) involvement in induction of differentiation in murine erythroleukemia (MEL) cells: (a) Effects of APAH , a N 8 -acetylspermidine deacetylase inhibitor, on differentiation induced by HMBA and its two deacetylated metabolites, NADAH and DAH , (b) influence of APAH on intracellular levels of HMBA and its deacetylated metabolites in HMBA treated MEL cells, (c) Ca 2+ mobilizing effects of HMBA, NADAH and DAH and (d) effect of APAH on HMBA induced changes in c- myc gene expression during differentiation. HMBA (5 mM) and DAH (2 mM) were equally effective in inducing MEL cell differentiation as measured by the amount of hemoglobin (Hb) produced, while NADAH (5 mM) was least effective. APAH (10–500 0μM) inhibited HMBA and NADAH induced differentiation without affecting DAH induced differentiation. APAH (500 μM) was shown to affect the deacetylation pathway for HMBA. There was a significant increase in intracellular NADAH levels and a decrease in DAH levels in MEL cells treated with both HMBA and APAH compared to HMBA alone (measured by LC/MS). This indicated that APAH inhibited the second deacetylation step, the conversion of NADAH to DAH but not the first, the conversion of HMBA to NADAH. Ca 2+ influx is necessary for HMBA induced MEL cell differentiation. BAPTA-AM (10 μM), a calcium chelator, inhibited HMBA induced Hb production while Tg (0.5 nM), the SERCA pump blocker, potentiated Hb production. 2-APB, a store operated channel (SOC) regulator, at higher concentrations (50,75 μM) prevented HMBA induced differentiation while at lower concentrations (5,10 μM) potentiated induced differentiation. DAH (0.5 mM), caused an immediate increase in [Ca 2+ ] i in MEL cells, while a slower response was seen with NADAH (3 mM). HMBA (5 mM) had the longest lag period (∼6 min) before it elevated [Ca 2+ ] i . APAH effectively prevented [Ca 2+ ] i increase caused by HMBA and NADAH but failed to alter DAH induced increase suggesting that DAH was the metabolite that raised [Ca 2+ ] i levels. Permeabilized cell assays demonstrated that DAH mobilized Ca 2+ from intracellular IP 3 sensitive stores in the ER. The identity of SOC for DAH induced Ca 2+ influx was inconclusive since 2-APB was not able to alter DAH induced Ca 2+ mobilizing responses. In addition to preventing HMBA induced MEL cell differentiation, APAH also inhibited the second phase of repression of c- myc gene expression, a hallmark of induced differentiation. In summary, the present study suggests the mechanism of action of HMBA requires the active involvement of a metabolite, DAH, in differentiation of hematopoietic cells.

Pages

184

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