The p53 tumor suppressor exerts (ejerce) anti-proliferative effects,

including growth (crecimiento) arrest(detención), apoptosis, and cell

senescence, in response to various types of stress. Inactivation of p53
function is critical to tumorigenesis. Mutations within(dentro) the p53
gene have(han) been(sido) well documented in more(mas) than(que)
half(mitad) of all human tumors. Accumulating evidence further
indicates that, in the cells that retain(retiene) wild(salvaje)-type p53,
other defects in the p53 pathway also play an important role in
tumorigenesis. Tight(firmemente) regulation of p53 is
imperative(impresindible) 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(se
piensa generalmente para implicar principalemente) 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(poco
grado) Lys320 by PCAF. The acetylation levels of p53 are significantly
enhanced(realzado) in vivo in response to almost every(a cada cas)i
type of stress, well correlated with its activation and stabilization
induced by stress. These(estos) 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(de
hecho), p53 acetylation has been shown to be (se han demostrado para
ser) critically important for the efficient recruitment(reclutamiento) of
these complexes to promoter regions and the activation of p53 target
genes in vivo.

Histone deacetylase complexes (HDACs) have emerged(emergido) as

notable components in regulating transcriptional activation.
Treatment(tratamiento) of cells with the HDACs inhibitor trichostatin A
(TSA) increases(incrementando) levels of acetylated p53 and
led(llevado) 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 (el trabajo
subsecuente) mammalian Sir2, a TSA-resistant, NAD-dependent histone
deacetylase that can both(ambos) deacetylate p53 and attenuate its
transcriptional activity. Sir2 co-localizes in PML nuclear bodies(cuerpos)
with p53 and was(era) structurally shown to undergo(experimente) a
conformational change when bound(límite) to acetylated p53. Further,
PML and oncogenic Ras can upregulate acetylated p53 levels in primary
fibroblasts. The novelty(novedad) of the Sir2 family of HDACs regulating
p53 function suggests(sugiere) an interesting link (acomplamiento)
between nicotinamide (vitamin B3), a Sir2 inhibitor, cellular metabolism,
and p53-mediated cellular responses(respuestas) to genotoxic stress.
Transgenic mice harboring(abrigar) 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(levadura) and C. elegans suggesting
that Sir2 may provide a possible link between p53 and mammalian
longevity.

My laboratory will focus (se enfocará) on demonstrating the precise role

of Sir2 in apoptosis and cell senescence, and to elucidate(aclare) this
novel p53 regulatory pathway in the stress response and to test new
drug for cancer therapy. To accomplish these goals (para lograr estas
metas), the following questions will be specifically addressed(las
preguntas siguientes serán tratadas: 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(desde entonces) 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(aislaremos)
naturally forming(formación) Sir2 containing(el contener) complexes from human cells by
using(el usar) the epitope-tagging(marcar con etiqueta) strategy. We will create a cell line
which stably expresses a Flag epitope-tagged full-length human Sir2. And isolate(aislante)
complexes containing(el contener) the epitope-tagged proteins. Any proteins specifically
co-purified with Sir2 will be analyzed by mass spectrometry to identify the novel proteins.
Each(cada) protein will be validated(validada) for its bona fide(autentico) interaction with
Sir2. This validation will include: i) in vitro use GST pull-down(desconexión)
assays(analisis) to investigate whether(sí) the interaction is direct, or through other
proteins{o atraves de otras proteinas); 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
Control of p53 transcriptional activity. DNA damage induces
phosphorylation, acetylation, stabilization of p53 polypeptides, which in
turn lead to an increase in the transactivation potential of p53.
Transcriptional activation of p53 can induce a variety of phenotypic
responses (e.g., growth arrest, cellular senescence, apoptosis)
depending on the cell type and nature of the cellular stress.
Deacetylation of p53 by Sir2 and/or PID/HDAC1 may be especially
important for downregulation of p53-dependent transcription once DNA-
damage response is complete. A, acetylation; P, phosphorylation; TFs,
transcription factors; TSA, trichostatin A.