BRCA1 localizes to discrete nuclear foci (dots) during S phase. Hydroxyurea-mediated DNA synthesis arrest of S phase MCF7 cells led to a loss of BRCA1 from these structures. Ultraviolet light, mitomycin C, or gamma irradiation produced a similar effect but with no concurrent arrest of DNA synthesis. BARD1 and Rad51, two proteins associated with the BRCA1 dots, behaved similarly. Loss of the BRCA1 foci was accompanied by a specific, dose-dependent change(s) in the state of BRCA1 phosphorylation. Three distinct DNA damaging agents preferentially induced this change in S phase. The S phase BRCA1 phosphorylation response to DNA damage occurred in cells lacking, respectively, two DNA damage-sensing protein kinases, DNA-PK and Atm, implying that neither plays a prime role in this process. Finally, after BRCA1 dot dispersal, BRCA1, BARD1, and Rad51 accumulated, focally, on PCNA+ replication structures, implying an interaction of BRCA1/BARD1/Rad51 containing complexes with damaged, replicating DNA. Taken together, the data imply that the BRCA1 S phase foci are dynamic physiological elements, responsive to DNA damage, and that BRCA1-containing multiprotein complexes participate in a replication checkpoint response.

These observations demonstrate increased chondrocyte apoptosis in OA cartilage. Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related.

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.

No abstract

Chondrocytes exposed to nitric oxide (NO) or antibody to Fas undergo cell death by apoptosis. This study examines structural and functional properties of chondrocyte-derived apoptotic bodies. In NO treated cartilage, the dense pericellular matrix that normally surrounds the cells is degraded and apoptotic bodies accumulate within and in the vicinity of the chondrocyte lacunae. Functional analysis shows that apoptotic bodies isolated from NO-treated chondrocytes or cartilage produce pyrophosphate. The levels of pyrophosphate produced by apoptotic bodies are increased by pretreatment of the chondrocytes with transforming growth factor ␤ and decreased by interleukin 1. Apoptotic bodies contain alkaline phosphatase and NTP pyrophosphohydrolase activities and can precipitate calcium. These results suggest that chondrocyte-derived apoptotic bodies express functional properties that may contribute to the pathologic cartilage calcification observed in aging and osteoarthritis.

The cellular role of the PML-containing nuclear bodies also known as ND10 or PODs remains elusive despite links to oncogenesis and viral replication. Although a potential role in transcription has been considered, direct evidence has been lacking. By developing a novel in vivo nucleic acid labeling approach, we demonstrate the existence of nascent RNA polymerase II transcripts within this nuclear body. In addition, PML and the transactivation cofactor, CREB binding protein (CBP), colocalize within the nucleus. Furthermore, we show that CBP in contrast to PML is distributed throughout the internal core of the structure. Collectively, these findings support a role for this nuclear body in transcriptional regulation.

No abstract

A central mechanism in apoptosis is the activation of proteases of the caspase (cysteine aspartases) family. Protease activation has also been implicated in necrosis, but its role in this cell death process and the identity of the proteases involved and their substrates, are unknown. Using human autoantibodies to well characterized cellular proteins as detecting probes in immunoblotting, we observed that a defined and somewhat similar set of nuclear proteins, including poly (ADP-ribose) polymerase (PARP) and DNA topoisomerase I (Topo I), were selectively cleaved during both apoptosis and necrosis of cultured cells induced by various stimuli. The resulting cleavage products were distinctively different in the two cell death pathways. In contrast to apoptosis, the cleavages of PARP and Topo I during necrosis were not blocked by the caspase inhibitor benzyloxycarbonylVal-Ala-Asp-fluoromethyl ketone (zVAD-fmk). These findings suggest that different proteases act in apoptosis and necrosis, and that although both cell death processes result in selective cleavage of almost identical cellular proteins, they can be distinguished immunochemically on the basis of their cleavage products.