Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition

Giammona, Lisa M.; Panuganti, Swapna; Kemper, Jan M.; Apostolidis, Pani A.; Lindsey, Stephan; Papoutsakis, Eleftherios T.; Miller, William M.

doi:10.1016/j.exphem.2009.08.004

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Volume 37, Issue 11 , Pages 1340-1352.e3, November 2009

Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD⁺ levels and SIRT inhibition

Lisa M. Giammona
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Ill., USA
- Dr. Giammona and Ms. Panuganti contributed equally to this work.
Swapna Panuganti
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Ill., USA
- Dr. Giammona and Ms. Panuganti contributed equally to this work.
Jan M. Kemper
- Master of Biotechnology Program, Northwestern University, Evanston, Ill., USA
Pani A. Apostolidis
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Ill., USA
- Delaware Biotechnology Institute, University of Delaware, Newark, Del., USA
Stephan Lindsey
- Delaware Biotechnology Institute, University of Delaware, Newark, Del., USA
- Department of Chemical Engineering, University of Delaware, Newark, Del., USA
Eleftherios T. Papoutsakis
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Ill., USA
- Delaware Biotechnology Institute, University of Delaware, Newark, Del., USA
- Department of Chemical Engineering, University of Delaware, Newark, Del., USA
William M. Miller
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Ill., USA
- Master of Biotechnology Program, Northwestern University, Evanston, Ill., USA
- The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Ill., USA
- Offprint requests to: William M. Miller, Ph.D., Northwestern University, 2145 Sheridan Road, Tech E136, Evanston, IL 60208

Received 19 June 2009; received in revised form 20 August 2009; accepted 20 August 2009. published online 27 August 2009.

Objective

Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood CD34⁺ cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy.

Materials and Methods

We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mobilized peripheral blood CD34⁺ cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD⁺ precursors. We tested additional inhibitors of the sirtuin (or SIRT) 1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets.

Results

NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure; 6.25 mM NIC increased NAD⁺ levels fivefold. Quinolinic acid increased NAD⁺ similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence.

Conclusion

NIC greatly increases Mk ploidy under a wide range of conditions without altering Mk morphology. Inhibition of SIRT1 and/or SIRT2 is primarily responsible for NIC effects on Mk maturation.