Polyamines are naturally occurring cellular polycations essential for cell growth and differentiation. This investigation focused on the quantitative analysis of polyamines and metabolites in mouse erythroleukemia (MEL) cells by mass spectrometry. Hexamethylene bisacetamide (HMBA) is a synthetic polyamine derivative known to induce differentiation of a variety of transformed cells such as MEL cells. A fast and sensitive quantitative method for HMBA and metabolites NADAH, DAH and AcHA was developed using atmosphere pressure chemical ionization mass spectrometry (APCI/MS) by flow injection analysis. Selected ion monitoring (SIM) mode was employed for the mass spectrometric detection and d 4 -DAH was used as the internal standard for quantitation. The intracellular concentrations of HMBA and metabolites were obtained in MEL cells treated with 5mM HMBA in the presence or absence of 500μM APAH, a potent N 8 -acetylspermidine deacetylase inhibitor. A significant increase in intracellular NADAH and decrease in DAH levels in MEL cells were observed in HMBA treatment in the presence of APAH. This result indicates that APAH inhibits the second deacetylation step in HMBA metabolism, the conversion of NADAH to DAH, but not the first deacetylation of HMBA to NADAH. Two histone deacetylase (HDAC) inhibitors including sodium butyrate (NaB) and m -carboxycinnamic acid bis-hydroxamide (CBHA) were also used as inducing agents for MEL cell differentiation. Both agents caused accumulation of hyperacetylated histone H4 and H2B in MEL cells at concentrations optimal for inducing differentiation, while neither HMBA nor APAH had detectable effect on the acetylation level of histones. A fast and sensitive method for five important polyamines including putrescine (PU), spermidine (SPD), spermine (SPM), N 1 -acetylspermidine (N 1 -AcSPD) and N 8 -acetylspermidine (N 8 -AcSPD) was also developed using APCI/MS by flow injection analysis. Selected reaction monitoring (SRM) mode was employed for the mass spectrometric detection and 1,7-diaminoheptane was used as the internal standard for quantitation. The intracellular polyamine concentrations was obtained in MEL cells treated with 5mM HMBA, 2mM NaB, 3μM CBHA and 500μM APAH respectively. A significant increase in N 8 -acetylspermidine levels was observed during 3hr to 4 days treatment with APAH. There was no change in N 8 -acetylspermidine levels in MEL cells treated either with NaB or CBHA. The results from the present study suggest APAH has a selective inhibitory effect on N 8 -acetylspermidine but not histone deacetylation. While HDAC inhibitors inhibit histone deacetylase but have no effect on N 8 -acetylspermidine deacetylation. In conclusion, despite the known similarities they share, the enzymes involved in the deacetylation process of N 8 -acetylspermidine and histones in MEL cells are different.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-3656 |
| Date | 01 January 2005 |
| Creators | Yuan, Jing |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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