The transcription coactivator and histone acetyltransferase CAMP response element–binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G(1)/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G(1)/S arrest in response to severe hypoxia. We found a general decrease in p16(INK4a) (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G(1)/S arrest. Furthermore, cell lines that underwent G(1)/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1alpha) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12-24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G(1)/S arrest, also did not show decreased HIF-1alpha, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3beta, which was the opposite pattern to that exhibited by the cell lines showing the G(1)/S arrest phenotype. Inhibition of GSK3beta by lithium chloride treatment of HeLa cells converted the HIF-1alpha, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1alpha, p16 and p27 was associated with both a hypoxia-induced G(1)/S arrest phenotype and increased GSK3beta activity.

ERCC1 protein expression using the FL297 antibody warrants further study as a potential prognostic marker in SCCHN.

1. Stone surface organic layers were investigated at five sites on small, acid streams in the Ashdown Forest, southern England. Sites differed in stream water pH (means 4.3-6.6) and some other physicochemical features.2. Organic layers at the stream bed surface differed between sites in structure and in the amount of organic carbon present. Algae were abundant at the sites with higher conductivity and pH, iron bacteria (mainly Leptothrix sp.) predominated in the iron-rich Broadstone stream, whereas the surface layer at the most acid site was predominantly a rather structureless organic film with few living organisms.3. Amounts of organic carbon on stones buried within the stream bed for 3 months were almost identical at all sites. When viewed with the scanning electron microscope (SEM). organic layers on buried stones were rather structureless with some amorphous, floceulent material. The paucity of microflora suggests that these layers may have been formed primarily by abiotic mechanisms.4. X-ray microanalysis of organic layers indicated that complexes of organic matter with the metals iron, aluminium and manganese were present.5. Laboratory experiments with seven species of invertebrates showed that all were able to remove and at least partially ingest organic layers and underlying sandstone. The weight of material removed was highest on microbially impoverished layers from the most acid site, and was substantially lower where algae were abundant.6. The role of stone surface organic layers in stream systems is discussed. They may be important sites of dissolved organic matter (DOM) uptake and transfer to the benthos. A major impact of acidity on running water could be through its effect on the structure and function of organic layers.