Apoptosis is one of the cellular responses activated in pre-cancerous cells that serve to delay the onset of malignancy. Proteins that induce apoptosis can be used to kill tumor cells. Nur77 is an orphan steroid receptor, whose induction was initially thought to cause apoptosis solely through transcriptional activation of its downstream genes. In some cancer cell lines, however, apoptotic-inducing agents like calcium ionophores and phorbol ester were shown to initiate apoptosis through translocation of Nur77 to mitochondria. There, Nur77 binds to Bcl-2 and converts Bcl-2 to a pro-apoptotic molecule by exposing its BH3 domain. A membrane spanning peptide containing the Nur77's Bcl-2 interacting domain can kill tumor cells. However, how Nur77 initiates apoptosis in a physiological setting was not clear. In thymocytes, T-cell receptor (TCR) inducible expression of Nur77 and its family member Nor-1 have long been known to be required for apoptosis of autoreactive thymocytes (negative selection). Recently, Nur77 and Nor-1 were found to translocate from nucleus to mitochondria in apoptotic thymocytes. Both Nur77 and Nor-1 can bind to Bcl-2 and expose the Bcl-2 BH3 domain in a T-cell receptor (TCR) signaling-dependent fashion. A similar exposure of the Bcl-2 BH3 epitope was also found in two in vivo models of negative selection. Nur77 can also bind to Bcl2A1, another member of the Bcl-2 family. Blocking Nur77 phosphorylation by protein kinase C inhibitors impedes Nur77 mitochondria translocation and apoptosis. Thus, Nur77/Nor-1 might mediate apoptosis of autoreactive thymocytes through the mitochondria pathway.
Aims i n this application are designed to test this hypothesis.
In aim 1, the nuclear versus mitochondrial apoptotic function of Nur77/Nor-1 in thymocytes will be studied. We will first assess the role of Nur77/Nor-1 phosphorylation in mitochondria translocation. The biological activities of Nur77/Nor-1 mutant proteins defective in mitochondria translocation, transcription activation or binding to Bcl-2 will then be studied using transgenic mice.
In aim 2, the in vivo contribution of Nur77/Nor-1 mitochondria translocation during negative selection will be examined. Transgenic mice expressing a Bcl-2 dominant-negative protein or mice with T-cell specific Nor-1 mutation at its phosphorylation site that affects its mitochondria translocation will be generated and characterized. T cell negative selection will be studied in these mice.
In aim 3, the pro-apoptotic function of Bcl-2 BH3 domains in vivo will be evaluated in T cell-specific transgenic mice over-expressing a Bcl-2 BH3 mutant protein and in Bcl-2 BH3 mutant knock-in mice that will be generated. Completion of these aims should lead to a significant advances in our understanding of T cell development, how Nur77/Nor-1 initiate apoptosis under normal physiological settings and how this pathway can be utilized to combat cancer.
Cancer arises by genetic changes that lead to resistance to cell death and out-of-control cell growth; inducing their intrinsic cell death pathways is one way to get rid of them. Nur77, a factor normally resides in the nuclear compartment, may induce cell death through conversion of an anti-apoptotic protein over- expressed in cancerous cells. Experiments proposed in this application seek to understand this process.
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