Quantitative analysis of the decay of immunoreactivity in stored prostate needle biopsy sections.
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Application of immunohistochemistry to assess the presence of prognostic tissue markers is used widely. The quantitation of these markers may be hampered by a time-related loss of antigenicity in formalin-fixed paraffin-embedded tissue stored on glass slides. Potential loss of immunohistochemical staining intensity was studied on prostatic needle biopsy sections stored for a maximum of 4 years with antibodies against p27kip1, CD-44s, MIB-1, and androgen receptor (AR). In benign tissue, the positive/total ratio for p27kip1 was determined, while CD-44s staining intensity was assessed semiquantitatively. For MIB-1 and AR, nuclear staining intensity was assessed using computed image analysis. An exponential and significant decay of immunoreactivity was seen for p27kip1, CD-44s, MIB-1, and AR, with half-lives of 587 days, 214 days, and 290 days for p27kip1, MIB-1, and AR, respectively. Immunohistochemical assessment of prognostic tissue markers on stored slides must be considered with care in research and clinical settings. (+info)
Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice.
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Mammalian nonhomologous DNA end joining employs Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and DNA ligase IV (Lig4). Herein, we show that Ku70 and Ku80 deficiency but not DNA-PKcs deficiency results in dramatically increased death of developing embryonic neurons in mice. The Ku-deficient phenotype is qualitatively similar to, but less severe than, that associated with XRCC4 and Lig4 deficiency. The lack of a neuronal death phenotype in DNA-PKcs-deficient embryos and the milder phenotype of Ku-deficient versus XRCC4- or Lig4-deficient embryos correlate with relative leakiness of residual end joining in these mutant backgrounds as assayed by a V(D)J recombination end joining assay. We conclude that normal development of the nervous system depends on the four evolutionarily conserved nonhomologous DNA end joining factors. (+info)
Fetal Alz-50 clone 1 (FAC1) protein interacts with the Myc-associated zinc finger protein (ZF87/MAZ) and alters its transcriptional activity.
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Transcription factors mediate their regulatory effects through interaction with DNA and numerous nuclear proteins. The fetal Alz-50 clone 1 (FAC1) protein, a novel DNA-binding protein with the capacity to repress transcription, is likely to function through a similar mechanism (1). Using the two-hybrid yeast screen, we have shown that FAC1 interacts with the myc-associated zinc finger protein (ZF87/MAZ). This association was confirmed in vitro with recombinant protein. The ZF87/MAZ interaction domain was mapped to the region containing a putative nuclear localization signal (NLS) and nuclear export sequence (NES) of FAC1, using deletion mutants of the FAC1 protein. FAC1, on the other hand, recognizes a conformational interface that includes the proline/alanine-rich domain of ZF87/MAZ and the first zinc finger. Cotransfection of NIH3T3 cells with ZF87/MAZ and a luciferase reporter containing the SV40 promoter and enhancer results in an increase in transcriptional activation, suggesting ZF87/MAZ is able to recognize its consensus binding site present in the SV40 promoter. Cotransfection with FAC1 reduces the level of ZF87/MAZ-induced activation of the SV40 promoter in a dose dependent manner. A mutant FAC1, lacking the ZF87/MAZ interaction domain, does not alter ZF87/MAZ activation of the SV40 promoter. These data demonstrate that interaction between FAC1 and ZF87/MAZ alters the transactivation capacity of ZF87/MAZ. By immunoblot analysis, FAC1 and ZF87/MAZ exhibit similar tissue distribution and co-localize to pathologic structures in Alzheimer's disease brain. Coexpression of FAC1 and ZF87/MAZ suggest that interaction of these two proteins will have biological implications for gene regulation in neurodegeneration. (+info)
DNA-dependent protein kinase activity correlates with Ku70 expression and radiation sensitivity in esophageal cancer cell lines.
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We investigated the relationship between DNA-dependent protein kinase (DNA-PK) activity and radiation sensitivity using 14 esophageal cancer cell lines, TE 1-14. DNA-PK activities differed significantly among the cell lines. The highest DNA-PK activity observed in TE-8 was more than two times higher than the lowest DNA-PK activity observed in TE-5. Significant correlation was observed between DNA-PK activity and D0 (r = 0.766; P = 0.0008). Western blots analysis revealed a significant correlation between DNA-PK activity and Ku70 expression, suggesting that the regulation in DNA-PK activity was associated with Ku70 expression. The data suggest that the measurement of DNA-PK activity and/or Ku70 expression may provide a useful way to predict radiation sensitivity. (+info)
Microvessel count, proliferating cell nuclear antigen and Ki-67 indices in gastric adenocarcinoma.
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The aim of the present study was to immunohistochemically investigate the prognostic value of neovascularization (expressed as microvessel count-MVC) and tumor cell proliferation (expressed as PCNA labeling index PLI and Ki-67 labeling index KLI) in gastric adenocarcinoma. Correlations with clinicopathologic features were also evaluated. Tumor specimens from 74 patients diagnosed as gastric adenocarcinoma were included in this study. Formalin fixed, paraffin embedded tissue sections stained immunohistochemically with F-VIII, PC10 and MIB-1 monoclonal antibodies. By ocular grid subdivided into 100 areas, number of microvessels and PC10, MIB-1 positive and negative cells were counted at x400 magnification. Chi-square test, Kaplan-Meier method and cox regression analysis were used for statistical analysis. The results showed that, MVC and PLI had a significant correlation with invasion and lymph node metastasis. The prognosis was significantly worse in patients with high MVC (>14 ) and with high PLI (>49%). However any relationship was not observed between KLI (38%) and clinicopathologic parameters, so KLI failed to predict the prognosis. Cox model showed that, MVC and PLI were independent prognostic variables. Ki-67 labeling index in gastric carcinomas has no prognostic relevance. However, the evaluation of microvessel count and proliferating cell nuclear antigen index in gastric carcinomas could be reliable indicators of prognosis. (+info)
Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors.
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Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription. (+info)
Ku recruits the XRCC4-ligase IV complex to DNA ends.
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Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway. The last two factors form a tight complex in cells. However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear. We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku. This interaction is stimulated once Ku binds to DNA ends. Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends. We further show that this recruitment is critical for efficient end-joining activity in vitro. Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation. Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells. These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases. We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required. (+info)
Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins.
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Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages. (+info)