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.

Polyamines are small, polycationic molecules required for growth and development and found in all living cells. In this study, the effects of two polyamine analogues, hexamethylene bisacetamide (HMBA), a differentiation inducer, and 7-[N-(3-aminopropyl)amino] heptan-2-one (APAH), an inhibitor of N8-acetylspermidine deacetylase, were studied using quantitative proteomics and stable-isotopes. Two new technologies, isotope-coded affinity tags (ICAT) and quantification in fragment spectra using isobaric stable isotope reagents (iTRAQ) were employed and compared. Quantitative results of these experiments showed few changes in the type and level of proteins detected in whole-cell extracts. Proteins from three populations of cells were studied, control (untreated), HMBA-treated, and HMBA plus APAH treated cells. Some of the proteins that were differentially expressed in response to these agents include pyruvate kinase (PK), lactate dehydrogenase (LDH), mini-chromosome maintenance protein 3 (MCM3), and poly-rC binding protein. The proteins PK and LDH have been reported as possible cancer markers. Histone protein levels were significantly reduced on HMBA treatment, and substantially recovered with the addition of APAH. This finding was very convincing in the iTRAQ work, but invisible to the ICAT experiment, because of the lack of cysteine residues required for quantification in the ICAT methodology. Two proteins were elevated in the HMBA-APAH experiment compared to the other two, heterogeneous nuclear ribonuclear protein C1/C2 (HNRP C1/C2) and ubiquitin. Considering their unique functions, the up-regulation of these proteins suggests the involvement of internal ribosome entry and protein degradation in response to APAH. The results of the two technologies, ICAT and iTRAQ, were found to overlap, but were partly complementary.