Section: Research

Jianyuan Luo, Ph.D.

Academic Role: Assistant Professor

Faculty Appointment(s) In:
   Cancer Biology

Other Affiliation(s):
   Interdisciplinary Graduate Program

P53 Acetylation/deacetylation Pathway and Cancer

The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress. Inactivation of p53 function is critical to tumorigenesis. Mutations within the p53 gene have been well documented in more than half of all human tumors. Accumulating evidence further indicates that, in the cells that retain wild-type p53, other defects in the p53 pathway also play an important role in tumorigenesis. Tight regulation of p53 is imperative for preventing tumorigenesis and maintaining normal cell growth. The precise mechanism by which p53 is activated by cellular stress is not completely understood; it is generally thought to involve mainly post-translational modifications of p53, including ubiquitination, phosphorylation and acetylation.

p53 is specifically acetylated at multiple lysine residues  of the C-terminal regulatory domain by CBP/p300, and to a lesser extent Lys320 by PCAF. The acetylation levels of p53 are significantly enhanced in vivo in response to almost every type of stress, well correlated with its activation and stabilization induced by stress. These acetylation sites of p53 are essential for its ubiquitination and subsequent degradation by Mdm2. They may also have a significant impact on protein-protein interactions between p53 and transcriptional co-activators such as CBP/p300 and PCAF.  Indeed, p53 acetylation has been shown to be critically important for the efficient recruitment of these complexes to promoter regions and the activation of p53 target genes in vivo.

Histone deacetylase complexes (HDACs) have emerged as notable components in regulating transcriptional activation. Treatment of cells with the HDACs inhibitor trichostatin A (TSA) increases levels of acetylated p53 and led to the identification of the adapter protein PID/MTA2, a component of the HDAC1 complex that can enhance HDAC1-mediated deacetylation of p53. Subsequent work has identified mammalian Sir2, a TSA-resistant, NAD-dependent histone deacetylase that can both deacetylate p53 and attenuate its transcriptional activity. Sir2 co-localizes in PML nuclear bodies with p53 and was structurally shown to undergo a conformational change when bound to acetylated p53. Further, PML and oncogenic Ras can upregulate acetylated p53 levels in primary fibroblasts. The novelty of the Sir2 family of HDACs regulating p53 function suggests an interesting link between nicotinamide (vitamin B₃), a Sir2 inhibitor, cellular metabolism, and p53-mediated cellular responses to genotoxic stress. Transgenic mice harboring an N-terminus p53 deletion mutant exhibit an early-ageing phenotype and Sir2 is involved in gene silencing and extension of life span in yeast and C. elegans suggesting that Sir2 may provide a possible link between p53 and mammalian longevity.

My laboratory will focus on demonstrating the precise role of Sir2 in apoptosis and cell senescence, and to elucidate this novel p53 regulatory pathway in the stress response and to test new drug for cancer therapy. To accomplish these goals, the following questions will be specifically addressed: 1) what is the role of Sir2 in regulation of cell senescence and apoptosis? 2) is there any novel regulatory factors for mammalian Sir2? 3) what is the effect on the cancer cells by inhibiting the p53 deacetylation pathways?

1. Examine the role for Sir2 in the regulation of cell senescence induced by oxidative stress

Sir2 is involved in gene silencing and extending life-span in yeast and C. elegans. Our previous results have provided evidence suggesting that mammalian Sir2 inactivates p53 by direct interactions and NAD-dependent deacetylation.  Since p53 activation is critical for oxidative stress-induced cell senescence, it will be very interesting to test the role of mammalian Sir2 in regulating p53-dependent cell senescence induced by oxidative stress.

2. Purification and identification of Sir2 associated protein complexes in human cells.

To identify novel Sir2 interacting proteins in mammalian cells, we will isolate naturally forming Sir2 containing complexes from human cells by using the epitope-tagging strategy. We will create a cell line which stably expresses a Flag epitope-tagged full-length human Sir2. And isolate complexes containing the epitope-tagged proteins. Any proteins specifically co-purified with Sir2 will be analyzed by mass spectrometry to identify the novel proteins. Each protein will be validated for its bona fide interaction with Sir2. This validation will include: i) in vitro use GST pull-down assays to investigate whether the interaction is direct, or through other proteins; ii) in vivo interaction use immunoprecipitation and Western blot analysis. After validation, candidate proteins will be further analyzed for their functional properties.

3. To test a novel drug for cancer therapy by inhibiting p53 deacetylation pathways

We have previously shown that p53 can be deacetylated by a PID/MTA2/HDAC1 complex, whose activity is completely abrogated in the presence of TSA. Recently, we found that nicotinamide has a strong inhibitory effect on Sir2 mediated deacetylation of p53 in vivo. When the wild-type p53 containing human lung carcinoma cells (H460) were treated by DNA damage reagent etoposide, and HDAC1 inhibitor TSA and Sir2 inhibitor nicotinamide, a super induction of p53 acetylation was induced. Thus, maximum induction of p53 acetylation requires inhibitors for both types of deacetylases (HDAC1 and Sir2) after DNA damage.

During past few years, numerous studies have demonstrated that the p53 acetylation can greatly enhance its transactivation activity, increase its stability and induce apoptosis. Here, we will use this three drug cocktail to test its effect on cancer cell line. Cell lines from different cancer such as HCT116, BL2, MCF-7 will be treated with different concentration to check the apoptosis effect on the cells. Meanwhile, normal human fibroblast such as IMR-90, WI-38 cells will be treated in same condition as control. The treated cells will be analyzed for 1) apoptotic cells (SubG1) by FACS analysis according to DNA content; 2) p53 acetylation level by the antibody specific to acetylated p53 and total p53 by anti p53(DO-1) antibody; 3) p53 target genes such as p21, BAX, Puma and Noxa expression level coordinate with apoptotic effect of the cells.

Office: LRB 422
Phone: 508-856-4437
E-mail: Jianyuan.Luo@umassmed.edu

More on Jianyuan Luo's Research Research | Figures | Publications | Biography View All Sections on One Page