No abstract

The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH 2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events. (Cancer Res 2005; 65(21): 9834-42)

The tissue and cellular distribution and regulation ofthe chromatin protein HI' has been examined in developing and adult mouse and in rat. The protein appears in specific cell types of solid tissues only when the cells have terminated their maturation. This was found for brain, retina, striated and cardiac muscle, and liver. In tissues that depend on hormones for their function and maintenance, the expression of HI' is dependent on the continued presence of the specific maintenance hormone. In regenerating rat liver the amount of HI0 decreases to one-third after the onset of DNA synthesis. The possible role of HI0 is discussed in light of these results.Proteins with properties similar or identical to those of the very lysine rich chromosomal protein Hi°have been described in several mammalian tissues and in tissue culture cells (1-16). The protein isolated from ox or mouse liver has been shown to consist of two subfractions that have identical molecular weights (25,000) but differ slightly in charge (2, 3). The two subfractions appear to have similar or identical amino acid compositions and peptide patterns. Recently it has been shown that Hi0 has extensive sequence homology with histone H5 from goose erythrocytes (4).Hi0 is of interest for several reasons. Its level in different tissues varies widely and it is predominantly found in tissues exhibiting little cellular proliferation (1,6,7,9,15,17). In earlier work, using indirect immunofluorescence with monospecific antibodies, we showed that Hi0 was present in many but not all fully mature postmitotic cells, and therefore that its distribution in different cells of the adult mouse is heterogeneous (15). These data suggest that Hi0 may play an important role in cell regulation.The concept that Hi0 is involved in developmental regulation is strengthened by the observations that in rat pancreas, liver, and spleen the protein appears after birth once cell proliferation in the organs has ceased (7,8,17). It has also been shown to decrease in regenerating rat pancreas and liver (5, 18).We report here studies on the regulation of Hi0 in developing mouse and in adult mouse and rat. The presence of Hi0 was shown to be correlated with cell maturation during development and liver regeneration. It was also found to be hormonally regulated in several glands in the adult. The results suggest that Hi0 may play a role in the maintenance of the mature differentiated state of some cells.MATERIALS AND METHODS Animals. Strain 129 male mice were used unless otherwise indicated. Fisher or Wistar male rats (both normal and hypophysectomized) were purchased from Iffa Credo (Paris, France). Hormones were injected either intraperitoneally or subcutaneously as described in the text or figure legends.Biochemical Methods. Lysine-rich histones were extracted from whole tissues with 5% perchloric acid as described (3). Proteins were analyzed by electrophoresis in polyacrylamide gels as described (10). After staining with Coomassie brilliant blue the gels were scanned at 590 nm and ...

Topoisomerase I (topo I) is required to unwind DNA during synthesis, and provides the unique target for camptothecin-derived chemotherapeutic agents, including Irinotecan and Topotecan. While these agents are highly effective anticancer agents, some tumors do not respond due to intrinsic or acquired resistance, a process that remains poorly understood. Because of treatment toxicity, there is interest in identifying cellular factors that regulate tumor sensitivity and might serve as predictive biomarkers of therapy sensitivity. Here we identify the serine kinase, protein kinase CK2, as a central regulator of topo I hyperphosphorylation and activity, and cellular sensitivity to camptothecin. In 9 cancer cell lines and 3 normal tissue-derived cell lines we observe a consistent correlation between CK2 levels and camptothecin responsiveness. Two other topo Itargeted serine kinases, protein kinase C and cyclin-dependent kinase1, do not show this correlation. Camptothecin-sensitive cancer cell lines display high CK2 activity, hyperphosphorylation of topo I, elevated topo I activity, and elevated phosphorylation-dependent complex formation between topo I and p14ARF, a topo I activator. Camptothecin-resistant cancer cell lines and normal cell lines display lower CK2 activity, lower topo I phosphorylation, lower topo I activity, and undetectable topo I/p14ARF complex formation. Experimental inhibition or activation of CK2 demonstrates that CK2 is necessary and sufficient for regulating these topo I properties and altering cellular responses to camptothecin. The results establish a cause and effect relationship between CK2 activity and camptothecin sensitivity, and suggest that CK2, topo I phosphorylation, or topo I/p14ARF complex formation could provide biomarkers of therapy responsive tumors. Figure S1), (2) Effect of CK2 activator on purified PKC, cdk1, and CK2 activities, and on endogenous PKC and cdk1 activity in H23 cells ( Figure S2), (3) Effect of CK2 activator on CK2α transcription ( Figure S3), (4) Levels of serine/threonine phosphatase activity in cell lines ( Figure S4), and (5) Topo-I phosphorylation levels in A549 and K562 cell lines ( Figure S5). The material can be accessed free of charge via the Internet at