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PubMed2004

Expression of DNA-PKcs and Ku86, but not Ku70, differs between lymphoid malignancies.

We have investigated the role of DNA-dependent protein kinase (DNA-PK) and related it to proliferation and maturation of different lymphoid malignancies. DNA-PK and Ki-67 protein content was investigated in tumour samples of lymphoid malignancies, obtained from patients with low- and high-grade lymphomas, acute lymphoblastic leukaemia and multiple myeloma. All patients were untreated before sampling. Normal bone marrow, reactive tonsillar tissue and ordinary lymph node tissue were used as controls. We show here that lymphoid malignancies display differences in DNA-PK protein expression. Low-grade lymphoma, appearing as chronic lymphocytic leukaemia (CLL) displayed a significantly lower frequency of cells staining positive for DNA-PKcs and Ku86, but surprisingly not for Ku70, compared with acute lymphoblastic leukaemia (ALL) cells. When material from individual CLL patients was investigated, cells from lymph nodes showed a higher frequency of positive cells with respect to all DNA-PK subunits, compared with CLL cells infiltrating the bone marrow. High-grade lymphoma lymph node samples showed an increased frequency of cells staining positive for DNA-PKcs, Ku86 and Ki-67 compared with lymph node samples from low-grade lymphoma patients. Again, no difference in the Ku70 levels between the two lymphoma entities was noted. In multiple myeloma, the frequency of cells with positive staining for DNA-PKcs was similar to that detected in ALL and high-grade lymphoma. We conclude that with the exception of multiple myeloma, expression of DNA-PK coincides with the degree of maturation of lymphoid malignancies. In low- and high-grade lymphoma, DNA-PK is associated with the proliferation rate.

Antigens, Nuclear↗
PubMed2004

The relationship between expression of Ku70 and survival in irradiated patients with endometrial carcinoma.

OBJECTIVE: To investigate the prognostic value of Ku70 protein expression in patients with endometrial cancer and to examine the correlation between Ku70 expression and established prognostic factors. METHODS: Ku70 immunoreactivity was determined by an immunohistochemical technique in a series of 81 patients with stages I-III primary endometrial carcinoma. The prognostic value of Ku70 expression was also studied in 74 patients who had been followed for at least 1 year or until death. RESULTS: The median percentage of Ku70 expression was 80%. The established clinicopathological prognostic factors for endometrial carcinoma were not associated with Ku70 expression. Although disease-free survival was significantly higher in patients with lower levels than median value of Ku70 (P < 0.05), overall survival was not different. In the univariate analysis, Ku70 expression did not show significant prognostic value for overall survival. The multivariate analysis also showed that Ku70 expression was not related to patient's outcome (P = 0.51, relative risk = 0.27, 95% confidence interval = 0.10-1.92). CONCLUSIONS: Our results suggest that Ku70 expression may not be a prognostic marker in endometrial carcinoma patients. However, disease-free survival was significantly higher in patients with low percentage of Ku70-positive tumor cells. More studies are needed to evaluate the correlation between Ku70 and survival in patients with endometrial cancer.

Adult↗
PubMed2005

Protein profiling and identification of modulators regulated by human papillomavirus 16 E7 oncogene in HaCaT keratinocytes by proteomics.

OBJECTIVES: Viral oncogenes E6 and E7 are selectively retained and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. This study was performed to identify proteins to be bound or modulated by high risk HPV E7 oncogene by using a proteomics. METHODS: HaCaT normal keratinocyte was prepared to establish a stable cell line expressing E7. The E7-affinity column was also prepared to obtain E7-interacting proteins. In order to search the target molecules modulated by E7 expression, we used 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight (MALDI/TOF) mass spectrometry. Pull down assay was also performed in order to confirm the E7-interacting proteins. RESULTS: We identified 28 spots that are modulated by E7 in HaCaT/E7 using 2-dimensional electrophoresis (2-DE) and MALDI/TOF mass spectrometry. Proteomics analyses showed that actin and leukocyte elastase inhibitor were down-regulated, whereas stress-induced phosphoprotein 1, CD2 binding protein 1, catalase, T-complex protein 1, Ku70-binding protein, heat shock 60 kDa protein 1, G1/S-specific cyclin E1 and peroxiredoxin 2 were up-regulated. Western blot revealed that heat shock 60 kDa protein, catalase and peroxiredoxin 2 were also up-regulated. Pull down assay also showed that leukocyte elastase inhibitor (LEI) and Ku70-binding protein were bound to the E7 oncoprotein. By using E7-affinity column and 2-DE/MALDI-TOF, 22 spots were found to interact with E7 recombinant protein. MG11-like proteins, livin inhibitor-of-apoptosis, protein serine kinase c17, CD2 binding protein 1, cyclin E1, TATA box binding protein-associated factor and uridine-cytidine kinase 2 were up-regulated by E7 oncogene and also bound to E7 oncoprotein. CONCLUSIONS: It is presumed that E7 can influence cell status by modulating the factors related to cell signaling, apoptosis and cell cycle regulation.

Antigens, Nuclear↗
PubMed1992

On the mechanisms of Ku protein binding to DNA.

The in vitro DNA-binding activity of Ku protein, a heterodimer of 70 and 86 kDa subunits, was studied using affinity-purified protein. Ku protein bound to different DNA probes and displayed a multiple-band pattern in band mobility shift assays. The protein-DNA complex formation was effectively blocked by different DNA competitors, indicating a non-sequence specific binding of Ku protein to DNA; no preference of binding of Ku protein to regulatory sequences derived from U1 snRNA, U6 snRNA or nucleolar protein p120 genes was observed. The number and size of the Ku protein-DNA complexes increased with increasing of the protein concentration and the size of DNA probe, suggesting that the protein accumulates on the DNA fragment until saturation of the binding sites. In UV-crosslinking experiments, the binding of Ku protein to DNA was shown to start with the 70 kDa subunit contacting free DNA ends.

Antigens, Nuclear↗
PubMed2000

Interaction of human Ku70 with TRF2.

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.

Animals↗
PubMed2003

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.

Antigens, Nuclear↗
PubMed1999

Somatostatin analogs stimulate DNA-dependent protein kinase activity in human gastric tumoral cell-line HGT1.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylates, in the presence of double-stranded DNA, several transcription-, replication- and repair -factors. Its interaction with the DNA-binding regulatory component Ku (p86-/p70-Ku) is required for stabilization and activity. We have previously shown that p86-Ku behaves as a specific receptor for the growth inhibitory tetradecapeptide, somatostatin. In this work, we investigate a possible regulation by somatostatin analogs, of DNA-PK activity in the human gastric tumoral HGT1/clone6 cell-line. We demonstrate that a 48 h-preincubation of cells with octreotide or RC-160, stimulates DNA-PK activity by 8 and 10 fold with ED50s of 1 and 0.1 nM, respectively. These stimulations appearing only after 3 h were inhibited by cycloheximide. They were not observed in a cell clone which was transfected by a cDNA encoding p86-Ku antisense. This study demonstrates the existence of a new somatostatin signaling pathway involving the stimulation of DNA-PK activity.

Antigens, Nuclear↗
PubMed2001

Recognition of cisplatin adducts by cellular proteins.

Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival.

Animals↗
PubMed2000

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine.

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.

Adult↗
PubMed2001

The impact energy metabolism and genome maintenance have on longevity and senescence: lessons from yeast to mammals.

The phenomenon that caloric restriction increases life span in a variety of species from yeast to mice has been the focus of much interest. Recent observations suggest that a protein important for heterochromatin formation, Sir2, is central for caloric restriction-induced longevity in lower organisms. Interestingly, Sir2 is also capable of repairing DNA double-strand breaks by nonhomologous end joining which may be important, along with proteins that repair breaks by recombinational repair, for minimizing the age-related deleterious effects of DNA damage induced by oxygen by-products of metabolism. I propose that competition between these two distinct functions could influence longevity and the onset of senescence. In addition, sequence and functional similarities between Sir2 and other chromatin metabolism proteins present the possibility that genetic components for longevity and senescence are conserved from yeast to mammals.

Aging↗
PubMed2003

DNA damage recognition and repair capacities in human naïve and memory T cells from peripheral blood of young and elderly subjects.

T cells accumulate genetic damage over time but nai;ve cells display higher genomic stability and longer lifespan as compared to memory cells. We found in nai;ve and memory T cells from young and elderly subjects that DNA damage in unirradiated cells is higher in memory than in nai;ve T cells, and is increased by radiation in both cell types. Repair of the radiation-induced DNA damage was much higher in nai;ve than in memory T cells from young subjects but null in both cell types from elderly subjects. Molecular mechanisms involved in DNA damage recognition and repair were analyzed in both cell subsets from young subjects. The intracellular distribution and amount of the DNA-dependent protein kinase (DNA-PK) complex components (ku 70, ku 80, DNA-PKcs), which are involved in the recognition and repair of DNA breaks caused by ionizing radiations, V(D)J recombination and isotype switching, was assessed in nai;ve and memory T cells from young subjects. While the expression of ku 70 and ku 80 was at comparable levels in both T cell subsets, DNA-PKcs, phosphorylated ku 80, and DNA-binding of ku 70/80 were mostly evident in nai;ve but negligible or absent in memory T cells. These findings may account for the higher genomic stability and longer lifespan of nai;ve as compared to memory human T cells from young subjects.

Adult↗
PubMedDate not supplied

Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction.

To provide an improved understanding of the molecular basis of the aging process, it is necessary to measure biological age on a tissue-specific basis. The role of DNA damage has emerged as a significant mechanism for determination of life span, and DNA repair genes and stress-response genes are also implicated in the aging process. In the present study, we investigated the changes of DNA-PK activity, especially Ku activity, in the various tissues including kidney, lung, testis and liver during aging and its correlation with mtHSP70 expression. We showed that the modulation of Ku activity during the aging process was highly tissue-specific as shown with highly impaired Ku activity in testis and unaffected Ku activity in liver with age, and the level of Ku70 or Ku80 was differentially expressed in each aging tissue. We found also that age-associated alteration of Ku70/80 was prevented or not prevented by caloric restriction (CR) in a tissue-specific manner. Age-related decline in Ku70 during the aging process was associated with the increase of mtHSP70, which could play a role as a predictive marker for aging related to Ku regulation, and CR retarded aging-induced mtHSP70.

Aging↗
PubMed1998

Cell proliferation and ku protein expression in ageing humans.

Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.

Adult↗
PubMed1996

Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates.

Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.

Animals↗
PubMed1999

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Antigens, Nuclear↗