Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220(NPAT) in human embryonic stem cells

Human embryonic stem (ES) cells have an expedited cell cycle ( approximately 15 h) due to an abbreviated G1 phase ( approximately 2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220(NPAT) and localization of HiNF-P/p220(NPAT) complexes with histone genes at Cajal body related subnuclear foci. Here we show that this 'S point' pathway is operative in situ in human ES cells (H9 cells; NIH-designated WA09). Immunofluorescence microscopy shows an increase in p220(NPAT) foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220(NPAT) foci is evident in S phase, the increase in the number of p220(NPAT) foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220(NPAT) at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF-P/p220(NPAT) pathway is rapidly established as ES cells emerge from mitosis and that p220(NPAT) is subsequently phosphorylated in situ. Our findings establish that the HiNF-P/p220(NPAT) gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication-linked histone genes and chromatin assembly to accommodate human stem cell self-renewal.     

Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines

The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G₀/G₁ phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level.     

Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self‐renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB‐dependent growth control. We investigated whether self‐renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220^NPAT/HiNF‐P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220^NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220^NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220^NPAT are principal cell cycle regulators that determine competency for self‐renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220^NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010. © 2009 Wiley‐Liss, Inc.