The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10 -17 with a neoR gene. ASPP2 ؊/؊ mice were not viable because of an early embryonic lethal event. Although ASPP2 ؉/؊ mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, ␥-irradiated 6-week-old ASPP2 ؉/؊ mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2 ؉/؉ mice. Primary thymocytes derived from ASPP2 ؉/؊ mice exhibited an attenuated apoptotic response to ␥-irradiation compared with ASPP2 ؉/؉ thymocytes. Additionally, ASPP2 ؉/؊ primary mouse embryonic fibroblasts demonstrated a defective G0/G1 cell cycle checkpoint after ␥-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.A poptosis-stimulating protein of p53-2 (ASPP2), also known as 53BP2L, encoded by TP53BP2 (1-3), enhances damage-induced apoptosis at least in part through a p53-mediated pathway (2, 4 -6). Depending on cell context and type of stress, ASPP2 levels increase via transcriptional or posttranslational mechanisms after cellular damage (4, 6). In addition to interacting with p53 (and family members) (5, 7), ASPP2 protein, and the 123-aa, amino-terminal, truncated splice isoform 53BP2/Bbp, also known as 53BP2S (3), interacts with several proteins involved in modulating apoptosis and cell growth, including Bcl-2, p65/RelA subunit of NF-B, Yesassociated protein-1, HCV core protein, APCL, and protein phosphatase-1 (8 -13). Additionally, ASPP2 is a direct E2F target gene, suggesting that it is a common link between the Rb/E2F and p53/p73 pathways (14 -16). ASPP2 expression is suppressed in many human cancers, and it has been associated with poor clinical outcome in patients with aggressive nonHodgkin's lymphoma treated with chemotherapy (2, 17-24). These findings suggest that ASPP2 is involved in important tumor suppression networks and the cellular damage response. Overexpression of ASPP2 or Bbp/53BP2S can suppress E1A and ras-mediated transformation of rat embryo fibroblasts (25,26), whereas attenuation of ASPP2 expression promotes clonogenic survival and inhibits apoptosis in cell culture (2, 4, 6) and promotes tumor formation in vivo (27). However, the mechanisms by which reduced ASPP2 expression enhances tumor formation in vivo remain to be elucidated.In this report, we targeted the ASPP2 allele in a mouse by using homologous recombination to explore the in vivo consequences of attenuated ASPP2 expression. We demonstrate that reduced ASPP2 express...

The tumor suppressor p53 plays a central role in the DNA damage response. p53 enhances base excision repair (BER), in part, through direct interaction with the repair complex. Mitochondrial DNA (mtDNA) is repaired by a mtBER pathway. Many colorectal cancers harbor mtDNA mutations that are associated with poor prognosis. In addition to modulating the apoptotic response, mitochondria-localized p53 also stimulates mtBER. However, the mechanisms by which p53 enhances colorectal cancer mtBER after stress remain unclear.To explore this, we used colorectal cancer cells isogenic for p53 (HCT116p53+/+ and HCT116p53À/À). p53+/+ cells more efficiently repaired H 2 O 2 damaged DNA in vivo as measured by semiquantitative mtDNA displacement loop PCR. Mitochondrial extracts from p53+/+ cells more efficiently stimulated 32 P-dCTP incorporation into a uracil-oligonucleotide. Recombinant p53 complemented p53À/À mitochondrial extract repair of uracil or 8-oxo-G-containing oligonucleotides. As a measure of DNA glycosylase activity, p53+/+ mitochondrial extracts more efficiently incised uracil or 8-oxo-G oligonucleotides, although recombinant p53 could not stimulate oligonucleotide incision. p53 did not influence mitochondrial apurinic/apyrimidinic endonuclease activity measured by incision of a tetrahydrofuran-oligonucleotide. p53+/+ mitochondrial extracts had higher DNA polymerase-; activity measured by 32 P-dCTP incorporation into a singlenucleotide gap oligonucleotide, and recombinant p53 complemented p53À/À mitochondrial extract DNA polymerase-; activity. mtDNA ligase activity was not affected by p53 status. p53 protein was detected in an inner mitochondrial membrane subfraction containing components of the mtBER complex. Our data suggest that an intact p53 pathway stimulates specific mtBER steps and provides mechanistic insight into the development of mtDNA mutations in colorectal cancer. (Cancer Res 2006; 66(7): 3485-94)

Both IgG and secretory IgA Abs in mucosal secretions have been implicated in blocking the earliest events in HIV-1 transit across epithelial barriers, although the mechanisms by which this occurs remain largely unknown. In this study, we report the production and characterization of a human rIgA2 mAb that carries the V regions of IgG1 b12, a potent and broadly neutralizing anti-gp120 Ab which has been shown to protect macaques against vaginal simian/HIV challenge. Monomeric, dimeric, polymeric, and secretory IgA2 derivatives of b12 reacted with gp120 and neutralized CCR5- and CXCR4-tropic strains of HIV-1 in vitro. With respect to the protective effects of these Abs at mucosal surfaces, we demonstrated that IgG1 b12 and IgA2 b12 inhibited the transfer of cell-free HIV-1 from ME-180 cells, a human cervical epithelial cell line, as well as Caco-2 cells, a human colonic epithelial cell line, to human PBMCs. Inhibition of viral transfer was due to the ability of b12 to block both viral attachment to and uptake by epithelial cells. These data demonstrate that IgG and IgA MAbs directed against a highly conserved epitope on gp120 can interfere with the earliest steps in HIV-1 transmission across mucosal surfaces, and reveal a possible mechanism by which b12 protects the vaginal mucosal against viral challenge in vivo.

The p53 pathway is a central mediator of the apoptotic response. ASPP2/ 53BP2L (apoptosis-stimulating protein of p53 2, also known as 53BP2L) enhances apoptosis through selective stimulation of p53 transactivation of proapoptotic target genes. Although the Rb/E2F pathway regulates ASPP2/ 53BP2L transcription, the complex mechanisms controlling ASPP2/ 53BP2L levels and function remain unknown. We now report that proteasomal degradation modulates ASPP2/ 53BP2L protein levels and apoptotic function. Treatment of cells with proteasomal inhibitors, including the clinically utilized proteasomal inhibitor bortezomib, increases ASPP2/ 53BP2L protein but not RNA levels. Likewise, anthracycline-based chemotherapy, which has multiple mechanisms of action, including proteasomal inhibition, increases ASPP2/ 53BP2L protein but not RNA levels. Proteasomal inhibition or anthracycline treatment increases ASPP2/ 53BP2L protein stability and half-life. Furthermore, the central region of the ASPP2/ 53BP2L protein is ubiquitinated as would be expected for a proteasomal substrate. More importantly, small interfering RNA knockdown of ASPP2/ 53BP2L levels attenuated bortezomib-induced apoptosis, and this effect was greater in wildtype p53 cells. Because elevated levels of ASPP2/ 53BP2L are proapoptotic, these results described an important new molecular mechanism that modulates the p53-ASPP2/ 53BP2L apoptotic pathway.

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