Project 3A: To examine whether delta133p53-expressing, CD28-restored and CD8+ T cells gain a cytotoxic activity on tumor cells in vitro, quantitative analyses of tumor cell survival and death will be done as in preivious studies. We will then use in vivo models in which human tumor xenografts are established in immunodeficient mice by orthotopic or subcutaneous injection of human tumor cells, followed by intravenous injection of delta133p53-expressing or control CD8+ T cells. The effects of delta133p53-expressing CD8+ T cells on tumor growth will be evaluated by quantitative image analysis, mouse survival curve analysis, and apoptosis assays on tissue sections. If successful, this study will be a significant step towards future clinical application of delta133p53 in cancer immunotherapy. Project 3B: We are currently accumulating data from irradiated human astrocytes in vitro. We have found that delta133p53 expression is diminished upon radiation-induced senescence of astrocytes, along with increased SASP (IL-6). Reconstitution of delta133p53 expression by lentiviral vector in astrocytes undergoing radiation-induced senescence has restored their proliferative potential and repressed SASP, and has upregulated RAD51 and enhanced DNA damage repair, probably due to gain-of-function of delta133p53. Our initial tissue section analysis showed that irradiated brain tissues obtained from two melanoma patients with brain metastasis had increased numbers of p16INK4A-positive senescent astrocytes, while non-irradiated brain tissues from a patient with the same disease and two non-disease controls did not. We will also apply our established expertise in astrocyte-neuron co-culture in vitro to investigating whether delta133p53-mediated regeneration from radiation-induced senescence and SASP can revert otherwise neurodegenerative astrocytes to neuroprotective ones, and whether such phenotypic reversion of astrocytes involves repressed IL-6 and increased NGF secretory signals from astrocytes to neurons. We are also performing experiments using chemotherapeutic drugs. The in vitro examination of the effect of delta133p53 on drug-induced astrocyte senescence and SASP and on astrocyte-mediated neuronal death and survival in co-culture, as well as examination of brain tissues from cancer patients who were treated with chemotherapeutic drugs, will be conducted. Drugs that cross the blood-brain-barrier (BBB) and are used for treatment of brain tumors will be examined for their direct effects on astrocytes and astrocyte-neuron interaction in culture. Project 3C: We plan to generate transgenic mice that will express delta133p53 in an inducible manner. In addition, as a complementary and alternative model to the humanized delta133p53 transgenic mice, we have found that human p53 knock-in (Hupki) mice express human delta133p53 protein. We have shown that the human delta133p53 is regulated via autophagic degradation as in human cells. In the Hupki mice, we plan to enhance endogenous delta133p53 expression by small molecule activators. We have recently found that temozolomide, a standard of care chemotherapeutic drug for glioblastoma has the ability to induce cellular senescence with downregulation of delta133p53, suggesting it as a chemotherapeutic drug. Project 3D: We examined primary fibroblasts isolated from HGPS patients and found that these disease fibroblasts also downregulate delta133p53 and upregulate p53beta upon replicative senescence. The shorter-than-normal replicative lifespan of these HGPS fibroblasts was significantly extended by increased expression of delta133p53 due to its dominant-negative inhibition of the senescence-inducing p53 target genes such as p21WAF1 and microRNA (miR)-34a. Delta133p53 also functioned to rescue the DNA repair defect characteristic of HGPS cells, marked by accumulated gamma-H2AX focus via a gain of function that transcriptionally activates the DNA repair factor RAD51. Thus, a therapeutic value of delta133p53 in premature aging diseases is supported by its ability to correct the cellular HGPS phenotypes such as shortened replicative lifespan and defective DNA repair by its dominant-negative and gain-of-function roles, respectively. We will examine the humanized delta133p53 transgenic and Hupki mice for their lifespan and aging-related pathologies to see whether delta133p53 has effects on physiological aging processes. These mice will also be crossed with mouse models of HG progeria to examine whether delta133p53 is able to rescue the disease-associated accelerated aging pathologies. Project 3E: Overexpression of delta133p53 extends the replicative lifespan of normal human cells but does not lead to cell immortalization, malignant transformation or chromosome abnormality. Normal human pluripotent stem cells, such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), express abundant levels of endogenous delta133p53. Enhanced expression of delta133p53 increases the efficiency of reprogramming from human fibroblasts to iPSCs that have normal chromosomes and form well-differentiated, benign teratomas, not malignant teratocarcinomas, which are induced by mutant p53. These delta133p53-inducible iPSC lines show lower rates of somatic mutations than iPSC under p53 knockdown. Whether these lower mutation rates are intrinsic to iPSC reprogramming or iPSC reprogramming is not by itself mutagenic, these data are strong evidence for no or minimal mutagenic activity of delta133p53. Unlike mutant p53, delta133p53 does not increase symmetric, self-renewing cell divisions in cancer stem cells, which are counterbalanced with asymmetric cell divisions by wild-type p53as. Lastly, higher levels of delta133p53 expression are correlated with better prognosis in colon and lung cancer patients. Thus our current data does not support the hypothesis that delta133p53 is oncogenic. Project 3F: We previously showed that p53beta was regulated by alternative mRNA splicing involving a splicing factor SRSF3. We have shown that the STUB1/CHIP E3 ubiquitin ligase has been identified as a critical delta133p53-interacting protein that regulates this autophagic degradation process and cellular senescence in these normal human cells. This mechanistic study of delta133p53 provides an experimental basis for mechanism-oriented screening of small molecules, rather than random screening, and suggests the use of known modulators of chaperone-assisted selective autophagy and STUB1/CHIP as controls in the screening. Towards development of p53 isoform-based therapies in both cancer and aging-associated diseases, we are seeking to identify small molecule compounds that upregulate the expression of delta133p53 protein, in particular, to those that cross the blood-brain-barrier (BBB) for astrocyte-mediated therapy in AD and radiation- and chemotherapy-induced brain damage. We are using a cell-based, robust high-throughput screening strategy at NCATS to detect upregulation of delta133p53. Our initial screening of the LOPAC library was performed and identified 35 hit compounds (2.7%), which show a specific, dose-dependent response. Several of these candidate activators of delta133p53 are known inhibitors of autophagy, as expected from the autophagic degradation of delta133p53. We will conduct the main screening of large-scale small molecule libraries. As in the initial LOPAC library screening, we expect that further hit compounds will include those that regulate chaperone-assisted autophagy and/or STUB1/CHIP. The activities of hit compounds will be first tested in our established in vitro assays. We will then confirm that they can upregulate delta133p53 in vivo in the various mouse models described above.
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