Mammalian cells have a remarkable diverse repertoire of response to genotoxic stress that damage DNA. Cellular responses to DNA damaging agents will initially exhibit gene induction, which is regulated by complex mechanism(s) and probably involves multiple signaling pathways. In this paper, we demonstrate that induction of ATF3 protein, a member of the ATF/CREB family of transcription factors, by ionizing radiation (IR) requires normal cellular p53 function. In contrast, induction of ATF3 after UV radiation (UV) or Methyl methanesulphonate (MMS) is independent of p53 status. Induction of ATF3 by DNA damage is rapid, transient, and through a transcriptional mechanism. The ATF3 promoter is induced by UV and MMS, but not by IR. In addition, ATF3 promoter can be activated by MEKK1, an upstream activator of the ERK and JNK kinase pathway, but not induced following p53 expression. Those results indicate that regulation of ATF3 induction after DNA damage utilizes both the p53-dependent andindependent pathways, and may also involve MAP kinase signaling pathways. Using the tetracyclineinducible system (tet-off), we have found that overexpression of ATF3 protein moderately suppresses cell growth. Interestingly, over-expression of ATF3 protein is able to slow down progression of cells from G1 to S phase, indicating that ATF3 protein might play a negative role in the control of cell cycle progression.

In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.

More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.

No abstract

Two-thirds of baboon sera demonstrated cytotoxicity to GT-KO PBMC. After GT-KO organ transplantation, if an elicited antibody response develops, it is likely to cause rapid graft rejection.

No abstract

Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled the study of incidence and cytotoxicity of antibodies of cynomolgus monkeys directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. To this aim, sera from 21 cynomolgus monkeys were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (WT) and GT-KO pigs. The sera were also tested for complement-dependent cytotoxicity to WT and GT-KO PBMC. Anti-WT IgM and IgG were found in 100% and 95%, respectively, and anti-GT-KO IgM and IgG in 76% and 66%, respectively, in the sera of the monkeys tested (P < 0.01). Whereas 100% of sera were cytotoxic to WT PBMC, only 76% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was significantly less (P < 0.01). Although the incidence and cytotoxicity of antibodies in monkey sera to GT-KO pig PBMC are significantly less than to WT PBMC, approximately three-quarters of the monkeys tested had cytotoxic antibodies to GT-KO PBMC. This incidence of cytotoxicity is significantly higher than that found in baboons and humans, suggesting the baboon may be an easier and possibly more suitable model to study antibody-mediated rejection of transplanted GT-KO pig organs and cells.