RADIOBIOLOGY DIVISION

12.RADIOBIOLOGY DIVISION

    Research in the Radiobiology Division is primarily focused on the two basic pathways in cancer: the RB pathway and the p53 pathway. In particular, the physiological meaning of phosphorylation of these proteins has been studied using many phospho-specific antibodies to recognize the phosphorylation sites on p53 and the RB proteins.
Apoptosis Induction by p53

    Studies in the Radiobiology Division showed previously that the switch between promoters of a G1-arrest gene, p21Waf1, and an apoptosis gene, p53AIP1 is regulated by phosphorylation of Ser46 on p53. It was subsequently shown that the Ser46-kinase is composed of several proteins and contains p53DINP1, a newly found protein which is induced by p53.
    Purification of Ser46-kinase was continued and it was found that casein kinase 2 is contained in this kinase complex. However, components of this kinase complex may be different when cells were stressed by UV. It was demonstrated that HIPK2 (homeodomain-interacting protein kinase-2) is activated by UV radiation and selectively phosphorylates p53 at Ser46, thus facilitating the CBP-mediated acetylation of p53 at Lys382, and promoting p53-dependent gene expression ⁽¹⁹⁹⁾. Accordingly, the kinase function of HIPK2 mediates the increased expression of p53 target genes, which results in growth arrest and enhancement of UV-induced apoptosis.
    On the other hand, p53AIP1 was characterized. Ectopic expression of p53AIP1 induced downregulation of mitochondrial membrane potential and release of cytochrome c from mitochondria in human cells ⁽²⁰⁰⁾. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and the Bcl-2 protein at the mitochondrial level. Overexpression of Bcl-2 blocked the down-regulation of the mitochondrial membrane potential and the proapoptotic activity of p53AIP1. These results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway.
Phosphorylation of Mdm2

    Mdm2 is a negative regulator of p53 which binds p53 and stimulates its degradation. The presence of multiple phosphorylation sites on it has been suggested, but the physiological function of these phosphorylation sites remained largely unknown.
    Although the function of cyclin G, a commonly induced p53 target, has remained elusive, it has now been shown that cyclin G forms a quarternary complex with phosphatase 2A (PP2A) as well as Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate human Mdm2 at Thr216 ⁽²⁰¹⁾. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at Thr216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Mdm2 at Ser166. Thus, these data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to stimulate the degradation of p53.
    In relation to the above mentioned finding, it was shown that Ser166 of human Mdm2 is phosphorylated by AKT (PKB) ⁽²⁰²⁾. AKT is well known to enhance cell survival and inhibit apoptosis.
    It was also shown that c-Abl binds and phosphorylates human Mdm2 at Tyr394, leading to inhibition of p53 degradation by Mdm2 ⁽²⁰³⁾. In response to DNA damage, c-Abl protects p53 by neutralizing the inhibitory effects of Mdm2. It defines a novel mechanism by which c-Abl activates p53.
Small Cell Lung Cancer Cell Lines with Neuron-like Processes

    Five of six human small cell lung cancer (SCLC) cell lines changed morphologically into cells with neuron-like processes on the extracellular matrix of PC-9 cells. It was observed that b-chains of tubulin and MAP-2 were expressed along the neuron-like processes of SCLC cell lines ⁽²⁰⁴⁾.




12.RADIOBIOLOGY DIVISION



	Research in the Radiobiology Division is primarily focused on the two basic pathways in cancer: the RB pathway and the p53 pathway. In particular, the physiological meaning of phosphorylation of these proteins has been studied using many phospho-specific antibodies to recognize the phosphorylation sites on p53 and the RB proteins. Apoptosis Induction by p53 Studies in the Radiobiology Division showed previously that the switch between promoters of a G1-arrest gene, p21Waf1, and an apoptosis gene, p53AIP1 is regulated by phosphorylation of Ser46 on p53. It was subsequently shown that the Ser46-kinase is composed of several proteins and contains p53DINP1, a newly found protein which is induced by p53. Purification of Ser46-kinase was continued and it was found that casein kinase 2 is contained in this kinase complex. However, components of this kinase complex may be different when cells were stressed by UV. It was demonstrated that HIPK2 (homeodomain-interacting protein kinase-2) is activated by UV radiation and selectively phosphorylates p53 at Ser46, thus facilitating the CBP-mediated acetylation of p53 at Lys382, and promoting p53-dependent gene expression ⁽¹⁹⁹⁾. Accordingly, the kinase function of HIPK2 mediates the increased expression of p53 target genes, which results in growth arrest and enhancement of UV-induced apoptosis. On the other hand, p53AIP1 was characterized. Ectopic expression of p53AIP1 induced downregulation of mitochondrial membrane potential and release of cytochrome c from mitochondria in human cells ⁽²⁰⁰⁾. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and the Bcl-2 protein at the mitochondrial level. Overexpression of Bcl-2 blocked the down-regulation of the mitochondrial membrane potential and the proapoptotic activity of p53AIP1. These results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway. Phosphorylation of Mdm2 Mdm2 is a negative regulator of p53 which binds p53 and stimulates its degradation. The presence of multiple phosphorylation sites on it has been suggested, but the physiological function of these phosphorylation sites remained largely unknown. Although the function of cyclin G, a commonly induced p53 target, has remained elusive, it has now been shown that cyclin G forms a quarternary complex with phosphatase 2A (PP2A) as well as Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate human Mdm2 at Thr216 ⁽²⁰¹⁾. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at Thr216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Mdm2 at Ser166. Thus, these data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to stimulate the degradation of p53. In relation to the above mentioned finding, it was shown that Ser166 of human Mdm2 is phosphorylated by AKT (PKB) ⁽²⁰²⁾. AKT is well known to enhance cell survival and inhibit apoptosis. It was also shown that c-Abl binds and phosphorylates human Mdm2 at Tyr394, leading to inhibition of p53 degradation by Mdm2 ⁽²⁰³⁾. In response to DNA damage, c-Abl protects p53 by neutralizing the inhibitory effects of Mdm2. It defines a novel mechanism by which c-Abl activates p53. Small Cell Lung Cancer Cell Lines with Neuron-like Processes Five of six human small cell lung cancer (SCLC) cell lines changed morphologically into cells with neuron-like processes on the extracellular matrix of PC-9 cells. It was observed that b-chains of tubulin and MAP-2 were expressed along the neuron-like processes of SCLC cell lines ⁽²⁰⁴⁾.