Robert L. Ochs

Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage

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.

Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis

OBJECTIVE To examine the occurrence of apoptosis in human osteoarthritis (OA) cartilage, and to determine its relationship to cartilage degradation. METHODS Knee cartilage was obtained from subjects at autopsy, from a tissue bank, and from OA patients undergoing total joint replacement surgery. Chondrocytes were isolated and the number of apoptotic cells was analyzed by flow cytometry. Apoptotic cells in cartilage sections were identified by the detection of DNA strand breaks. Electron microscopy was applied to demonstrate morphologic changes, and Safranin O staining was performed to analyze the relationship between apoptosis and proteoglycan depletion. RESULTS Flow cytometry on cell suspensions prepared from collagenase digests of cartilage showed that approximately 22.3% of OA chondrocytes and 4.8% of normal chondrocytes were undergoing apoptosis. Staining of cartilage sections demonstrated the presence of apoptotic cells in the superficial and middle zones. Cartilage areas that contained apoptotic cells showed proteoglycan depletion, and the number of apoptotic cells was significantly correlated with the OA grade. CONCLUSION These observations demonstrate increased chondrocyte apoptosis in OA cartilage. Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related.

Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes

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 benzyloxycarbonyl-Val-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.

Nuclear bodies (NBs): A newly “rediscovered” organelle

Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.

Nuclear apoptotic changes: An overview

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post‐translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components. J. Cell. Biochem. 82: 634–646, 2001.

Autoantibodies in Interstitial Cystitis

We have identified and partially characterized autoantibodies from the sera of patients with interstitial cystitis. Our characterization included initial screening by antinuclear antibody testing on human HEp-2 cell substrate and mouse kidney/stomach tissue substrate, titering and subtyping of positive sera, and Western blotting to identify target autoantigens. Of 96 interstitial cystitis patients 35 (36%) were positive for antinuclear antibodies at titers of 1/40 or greater. Among the antinuclear antibody patterns observed 24 were dense fine nuclear speckles, 7 were nucleolar, 3 were mitochondrial and 1 was coarse nuclear speckles. All but 4 of the antinuclear antibody positive sera were exclusively of the IgG class. As determined by unique antinuclear antibody staining patterns and by specificities on Western blots, interstitial cystitis autoantibodies appear to recognize novel autoantigens not previously described in patients with systemic autoimmune diseases, such as lupus, scleroderma and Sjögrens syndrome.

Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis.

OBJECTIVE To examine the pathologic changes in meniscus tissue during experimental osteoarthritis (OA) and to determine the relationship between nitric oxide (NO) synthesis, apoptosis, and meniscus degradation. METHODS OA was induced in rabbits by anterior cruciate ligament (ACL) transection. Knees were harvested after 9 weeks and assessed for OA severity. Menisci were subjected to histologic, immunohistochemical, and electron microscopic analyses for the presence of nitrotyrosine and apoptosis. Menisci were also cultured for analysis of NO production. RESULTS All menisci from joints with ACL transection demonstrated degenerative changes. A high number of apoptotic cells was present in the medial part of menisci, which contains chondrocytic cells. Menisci from nonoperated contralateral knees contained only small numbers of cells in apoptosis. Conditioned media from meniscus cultures contained similarly elevated levels of nitrite as cartilage cultures from the same arthritic knees. Nitrotyrosine immunoreactivity, an indicator of in vivo NO production, was prominent in menisci from knees with ACL transection. In addition, menisci from normal knees produced high levels of NO in response to in vitro stimulation with interleukin-1beta or lipopolysaccharide. CONCLUSION These observations suggest that pathologic changes in menisci are a regular feature of experimentally induced OA and are associated with NO production and meniscus cell apoptosis.

Regulation of YKL-40 production by human articular chondrocytes

OBJECTIVE YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. METHODS Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. RESULTS YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. CONCLUSION The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein.

METHODS USED TO STUDY STRUCTURE AND FUNCTION OF THE NUCLEOLUS

Publisher Summary The main function of the nucleolus is to synthesize the ribosomal RNA (rRNA) that, together with protein, becomes the building block of cytoplasmic ribosomes. Because the synthesis and processing of precursor preribosomal RNA seem to occur in a more or less vectorial fashion from the central portion of the nucleolus to its periphery and because the nucleolus is essentially a large ribonucleoprotein complex whose structure and morphology are directly related to its synthetic activity, the relationship between nucleolar structure and function is intimately linked and could perhaps be regarded as inseparable. The chapter presents a diagram that illustrates the main nucleolar compartments of fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). It also summarizes the relationship between nucleolus structure and function, including nucleolar proteins localized to each structural compartment. Methods on nucleolus research are also discussed; these methods are successfully employed for their reproducibility, simplicity, and ease of use.

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.