PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET

Pharmacological targeting of mutant p53 family members

The tumor suppressor p53 serves as a guardian of the genome and functions mainly as a transcription factor. In response to various stress signals p53 binds to specific DNA sequence motifs and regulates transcription of a large group of target genes involved in cellular processes such as cell cycle arrest, senescence and apoptosis. Inactivation of p53 is critical for the formation of most tumors. Around half of all human cancers carry mutations in the p53 gene (TP53) and mutant p53-harbouring tumors often show increased resistance to conventional chemotherapy. Therefore, pharmacological restoration of wild type function to mutant p53 is a promising strategy for novel cancer therapy. We have identified a low molecular weight compound, STIMA-1, that selectively targets tumor cells in a mutant p53- dependent manner. STIMA-1 contains a reactive double bond that can potentially participate in Michael addition reactions and may restore the tumor suppressive function to mutant p53 by affecting its redox status. Several other small molecules that reactivate mutant p53 have been identified in our group. PRIMA-1 and its more potent analog PRIMA-1(MET) (also denoted APR-246) both induce p53 target genes and mutant p53-dependent apoptosis in human tumor cells. PRIMA-1 and PRIMA-1(MET) are under physiological conditions converted to MQ that binds covalently to the p53 core domain and this modification per se is sufficient to endow mutant p53 with pro-apoptotic properties. To further explore the effects of PRIMA-1 and its analogs on tumor cells we analyzed the subcellular distribution pattern of several proteins upon drug treatment. We found that PRIMA- 1 and PRIMA-1(MET), but not PRIMA-Dead (a PRIMA-1 analog that is unable to induce apoptosis) induced nucleolar accumulation of mutant p53. In addition, PRIMA-1(MET) induced the levels of heat shock protein (Hsp) 70 and a redistribution of the PML nuclear body-associated proteins CBP, PML, Hsp70, and the Epstein- Barr virus encoded protein EBNA-5 to nucleoli. Our results suggest that relocation of mutant p53 and/or PML nuclear body-associated proteins to nucleoli may play a role in PRIMA-1(MET)-induced apoptosis. Since p53 and its family members p63 and p73 share high sequence and structural homology, we examined if PRIMA-1(MET) also affects mutant p63 and p73. We found that PRIMA-1(MET) restores wild type activity to some mutant forms of p63 and p73. PRIMA-1(MET) enhanced mutant p63 DNA binding, and induction of target gene expression and apoptosis in human tumor cells in a mutant p63/p73 dependent manner. PRIMA-1(MET) also induced a redistribution of mutant p63 to PML nuclear bodies and to nucleoli. Our data indicate that PRIMA-1(MET) exerts its effects through a common mechanism for all three p53 family members, presumably involving homologous structural domains in the three proteins. A better understanding of the exact molecular mechanisms of p53-targeting compounds is highly relevant for further drug optimization and the design of novel compounds with improved target selectivity and potency. The effect of PRIMA- 1(MET) on mutant p63 also raises the possibility of pharmacological rescue of p63 mutants in human developmental disorders caused by mutations in p63

Grothendieck Rings and Motivic Integration

This thesis consists of three parts: In Part I we study the Burnside ring of the finite group G. This ring has a natural structure of a lambda-ring. However, a priori the images of the G-set S under the lambda-operations can only be computed recursively. We establish an explicit formula, expressing these images as linear combination of classes of G-sets. This formula is derived in two ways: First we give a proof that uses the theory of representation rings in an essential way. We then give an alternative, more intrinsic, proof. This second proof is joint work with Serge Bouc. In Part II we establish a formula for the classes of certain tori in the Grothendieck ring of varieties, in terms of its lambda-structure. More explicitly, we will see that if L* is the torus of invertible elements in the n-dimensional separable k-algebra L, then the class of L* can be expressed as an alternating sum of the images of the spectrum of L under the lambda-operations, multiplied by powers of the Lefschetz class. This formula is suggested from the cohomology of the torus, illustrating a heuristic method that can be used in other situations. To prove the formula will require some rather explicit calculations in the Grothendieck ring. To be able to make these we introduce a homomorphism from the Burnside ring of the absolute Galois group of k, to the Grothendieck ring of varieties over k. In the process we obtain some information about the structure of the subring generated by zero-dimensional varieties. In Part III we give a version of geometric motivic integration that specializes to p-adic integration via point counting. This has been done before for stable sets; we extend this to more general sets. The main problem in doing this is that it requires to take limits, hence the measure will have to take values in a completion of the localized Grothendieck ring of varieties. The standard choice is to complete with respect to the dimension filtration. However, since the point counting homomorphism is not continuous with respect to this topology we have to use a stronger one. We thus begin by defining this stronger topology; we will then see that many of the standard constructions of geometric motivic integration work also in this setting. Using this theory, we are then able to give a geometric explanation of the behavior of certain p-adic integrals, by computing the corresponding motivic integrals