Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins. (1/1378)

The PML protein is associated to nuclear bodies (NBs) whose functions are as yet unknown. PML and two other NBs-associated proteins, Sp100 And ISG20 are directly induced by interferons (IFN). PML and Sp100 proteins are covalently linked to SUMO-1, and ubiquitin-like peptide. PML NBs are disorganized in acute promyelocytic leukemia and during several DNA virus infections. In particular, the HSV-1 ICP0 protein is known to delocalize PML from NBs. Thus, NBs could play an important role in oncogenesis, IFN response and viral infections. Here, we show that HSV-1 induced PML protein degradation without altering its mRNA level. This degradation was time- and multiplicity of infection-dependent. Sp100 protein was also degraded, while another SUMO-1 conjugated protein, RanGAP1 and the IFN-induced protein kinase PKR were not. The proteasome inhibitor MG132 abrogated the HSV-1-induced PML and Sp100 degradation and partially restored their NB-localization. HSV-1 induced PML and Sp100 degradation constitutes a new example of viral inactivation of IFN target gene products.  (+info)

Apoptosis during breast carcinoma progression. (2/1378)

The purpose of this study was to investigate apoptosis, proliferation, and the expression of apoptosis-influencing proteins bcl-2 and bax and estrogen and progesterone receptors during breast carcinoma progression. The material consisted of 53 paired breast carcinoma samples representing primary and recurrent tumors and 24 control samples. The recurrent sample was located either in the breast scar tissue or at a distant metastatic site. Apoptosis was detected both morphologically and by 3' end labeling of fragmented DNA. Cell proliferation was evaluated immunohistochemically by the MIB index. The expressions of bcl-2, bax, and estrogen and progesterone receptors were studied immunohistochemically. There was a significant increase in the extent of apoptosis and proliferation in recurrent tumors compared to the primary lesions (P = 0.015 and P = 0.038, respectively). In primary tumors with an apoptotic index of >0.50%, the survival of the patients was significantly shorter (P = 0.015). In cases with a significant increase in apoptosis or proliferation in the recurrent tumor, the survival of the patients was significantly shorter (P = 0.009 and P = 0.003, respectively). Of the variables analyzed, bcl-2 expression and a positive estrogen receptor status were significantly associated with a low extent of apoptosis (P = 0.010 and P = 0.042, respectively). Their changes were parallel to the changes in apoptosis during tumor progression, although the associations did not reach statistical significance. The results show that increased apoptosis is associated with a worse prognosis in breast carcinoma. A significant increase in apoptosis in recurrent breast carcinoma lesions predicts a worse clinical outcome.  (+info)

Yeast and human genes that affect the Escherichia coli SOS response. (3/1378)

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the Escherichia coli SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g., RAD51 and HHT1 as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.  (+info)

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. (4/1378)

Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. KEYWORDS: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation  (+info)

OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication. (5/1378)

Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of approximately 92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication.  (+info)

Androgen receptor expression in male breast carcinoma: lack of clinicopathological association. (6/1378)

Androgen receptor (AR) expression was retrospectively analysed in 47 primary male breast carcinomas (MBCs) using a monoclonal antibody on formalin-fixed, paraffin-embedded tissues. AR immunopositivity was detected in 16 out of 47 (34%) cases. No association was found with patient age, tumour stage, progesterone receptor (PGR) or p53 protein expression. Well-differentiated MBCs tended to be AR positive more often than poorly differentiated ones (P = 0.08). A negative association was found between ARs and cell proliferative activity: MIB-1 scores were higher (25.4%) in AR-negative than in AR-positive cases (21.11%; P = 0.04). A strong positive association (P = 0.0001) was found between ARs and oestrogen receptors (ERs). In univariate analysis, ARs (as well as ERs and PGRs) were not correlated with overall survival; tumour histological grade (P = 0.02), size (P = 0.01), p53 expression (P = 0.0008) and MIB-1 scores (P = 0.0003) had strong prognostic value. In multivariate survival analysis, only p53 expression (P = 0.002) and histological grade (P = 0.02) retained independent prognostic significance. In conclusion, the lack of association between AR and most clinicopathological features and survival, together with the absence of prognostic value for ER/PGR status, suggest that MBCs are biologically different from female breast carcinomas and make it questionable to use antihormonal therapy for patients with MBC.  (+info)

The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA. (7/1378)

The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells. DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460). Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process. It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity. Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA. We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini. When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation. The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini.  (+info)

Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice. (8/1378)

NuMA is a nuclear matrix protein in interphase and relocates to the spindle poles in mitotis. Different NuMA constructs, in which either N- or C-terminal domains were deleted, and the full-length construct were expressed in Escherichia coli, and the NuMA polypeptides were purified to homogeneity and allowed to assemble in vitro. Electron microscopy showed that NuMA can build multiarm oligomers by interaction of the C-terminal globular domains. Each arm of the oligomer corresponds to a NuMA dimer. Oligomers with up to 10 or 12 arms have been observed for both full-length NuMA and for constructs that still contain the proximal part of the C-terminal tail domain. Other results from this laboratory have shown that transient overexpression of NuMA in HeLa cells induces a nuclear scaffold with a quasi-hexagonal organization that can fill the nuclei. Here we show that computer modelling of the three-dimensional packing of NuMA into such scaffolds can explain the different spacing of the hexagons seen when constructs with different coiled-coil lengths are used. Thus, the 12 arm oligomer, for which we have in vitro evidence, may be the structural unit from which the nuclear scaffold in transfected cells is built.  (+info)

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