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Animals↗
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A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.
XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.
Animals↗
Involvement of DNA-dependent protein kinase in regulation of the mitochondrial heat shock proteins.
Since DNA-dependent protein kinase (DNA-PK) has been known to play a protective role against drug-induced apoptosis, the role of DNA-PK in the regulation of mitochondrial heat shock proteins by anticancer drugs was examined. The levels of basal and drug-induced mitochondrial heat shock proteins of drug-sensitive parental cells were higher than those of multidrug-resistant (MDR) cells. We also demonstrated that the development of MDR might be correlated with the increased expression of Ku-subunit of DNA-PK and concurrent down-regulation of mitochondrial heat shock proteins. The basal mtHsp70 and Hsp60 levels of Ku70(-/-) cells, which were known to be sensitive to anticancer drugs, were higher than those of parental MEF cells, but conversely these mitochondrial heat shock proteins of R7080-6 cells over-expressing both Ku70 and Ku80 were lower than those of parental Rat-1 cells. Also, the mtHsp70 and Hsp60 levels of DNA-PKcs-deficient SCID cells were higher than those of parental CB-17 cells. Our results suggest the possibility that mitochondrial heat shock protein may be one of determinants of drug sensitivity and could be regulated by DNA-PK activity.
Animals↗
Constitutively active mutant D816VKit induces megakayocyte and mast cell differentiation of early haemopoietic cells from murine foetal liver.
Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF.
Antigens, Nuclear↗
Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.
Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.
Animals↗
Human satellite 3 (HS3) binding protein from the nuclear matrix: isolation and binding properties.
Satellite DNA (satDNA) is the main component of residual DNA in nuclear matrix (NM) preparations. Gel mobility shift assay (GMSA) revealed specific human satellite 3 (HS3) binding activity in NM extracts. An HS3 binding protein was purified using diethylaminoethyl (DEAE)-cellulose and preparative GMSA. The binding was specific, although other satDNA fragments compete to some extent for the binding. DNase I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with periodicity of about 10 bp, mostly inside an AT island. Polyclonal antibodies (AB) were raised against HS3-protein complexes cut from the preparative GMSA gel. On immunoblots, AB recognise a protein, which is not lamin, with apparent molecular mass 70 kDa, the same as revealed by purification (p70). In in situ nuclear matrix preparations combined immunofluorescence (AB) and fluorescent in situ hybridisation (HS3) shows that HS3 and p70 areas correspond to each other. The localisation of this protein detected with AB in interphase nuclei coincides with the heterochromatic regions which surround nucleoli in correspondence with the known HS3 position in the nuclei.
Antibodies↗
Ku protein in human T and B lymphocytes: full length functional form and signs of degradation.
DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks. Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK. In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length. Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx. 70 kDa.
Antigens, Nuclear↗
Anti-Ku antibodies in connective tissue diseases. Report of three cases.
Three patients are described with clinical features of connective tissue diseases, namely, dermatomyositis, progressive systemic scleroderma, and systemic lupus erythematosus. In two patients the symptoms of disease overlapped. The rare anti-Ku antibody was found in all of them. Anti-Ku antibody characterizes those patients, with polymyositis-scleroderma overlap syndromes who have a good prognosis. One of our patients, who also had severe anti-Sm-positive systemic lupus erythematosus, seems to be an exception.
Adult↗
Is DNA repair compromised in Alzheimer's disease?
Mammalian cells utilize multiple mechanisms to repair DNA damage that occurs during normal cellular respiration and in response to genotoxic stress. This study sought to determine if chronic oxidative stress proposed to occur during Alzheimer's disease alters the expression or activity of DNA double-strand break repair or base excision repair proteins. Double-strand break repair requires DNA-dependent protein kinase, composed of a catalytic subunit, DNA-PKcs, and a regulatory component, Ku. Ku DNA binding activity was reduced in extracts of postmortem AD midfrontal cortex, but was not significantly different from the age-matched controls. Decreased Ku DNA binding correlated with reduced protein levels of Ku subunits, DNA-PKcs, and poly(ADP-ribose) polymerase-1. Expression of the base excision repair enzyme Ref-1, however, was significantly increased in AD extracts compared to controls. Ku DNA binding and DNA-PK protein levels in the AD cases correlated significantly with synaptophysin immunoreactivity, which is a measure of synaptic loss, a major correlate of cognitive deficits in AD. Immunohistochemical analysis suggested that DNA-PK protein levels reflected both number of neurons and regulation of cellular expression.
Adult↗
Genetic factors in immunity and aging.
Maximum life span is controlled by genes that regulate molecular mechanisms accounting for the synchrony of structural and functional changes in different cells and tissues of each member of a given species. The role of immune response genes was investigated in aging mice genetically selected for high (H) or low (L) antibody response (Biozzi mice). Results from genetic selection of over 1000 mice showed that genes expressed in the immune system affect life span and diseases. In most cases, the life span is longer in H than in L mice whereas the lymphoma incidence is remarkably higher in L than in H mice. Since DNA repair capacity is a property positively correlated with the maximum life span in several mammalian species, DNA repair was studied by use of hydroxyurea, a cell-synchronizing agent, and found to take place in irradiated human PBMC from young and, to a lesser extent, from adult subjects. Conversely, no repair was detected in irradiated PBMC from elderly subjects. DNA damage recognition and repair pathways involve several nuclear proteins, as double strand breaks are firstly recognized by proteins displaying helicase activity, such as ku 70/80, and then repair is carried out under the control of other proteins. Radiation-induced expression of activated ku(70/80) proteins, in terms of DNA-binding, was found in PBMC from young-adults but not from elderly subjects. Maintenance of DNA integrity is fundamental for normal immune functions, as suggested by the lack of V(D)J recombination in lymphocytes of knock-out mice deficient in ku 70 or ku 80 protein. However, whether the link between genetic factors and life span is mediated by the performance of the immune system remains to be demonstrated.
Adult↗
The activity of the DNA-dependent protein kinase (DNA-PK) complex is determinant in the cellular response to nitrogen mustards.
The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.
Animals↗
Regulation of the DNA-dependent protein kinase (DNA-PK) activity in eukaryotic cells.
The DNA-dependent protein kinase (DNA-PK) is a trimeric nuclear serine/threonine protein kinase consisting of a large catalytic sub-unit and the Ku heterodimer that regulates kinase activity by its association with DNA. DNA-PK is a major component of the DNA double strand break repair apparatus, and cells deficient in one of its component are hypersensitive to ionizing radiation. DNA-PK is also required to lymphoid V(D)J recombination and its absence confers in mice a severe combined immunodeficiency phenotype. The purpose of this review is to summarize the current knowledge on the mechanisms that contribute to regulate DNA-PK activity in vivo or in vitro and relates them to the role of DNA-PK in cellular functions. Finally, the studies devoted to drug-inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.
Animals↗
Regulation of CD34 transcription by Sp1 requires sites upstream and downstream of the transcription start site.
CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on fully differentiated cells in the peripheral blood. To better understand the molecular regulation of early hematopoiesis, we are elucidating the mechanisms of CD34 transcriptional regulation. By deletion analysis we identify a 39-bp element in the proximal region of murine CD34 promoter that is critical for promoter activity. Electromobility shift assays indicate that nuclear proteins of hematopoietic cells bind to this domain; however, the presence of this binding activity does not correlate directly with CD34 expression.Using methylation interference, the DNA binding site for this activity was localized to four guanine residues within the GGGGTCGG sequence from -48 to -54 bp. When the four contact guanines were mutated, both protein binding and promoter activity were abolished. Although this sequence does not contain a standard consensus for Sp1, this transcription factor binds specifically to the 39-bp region and stimulates promoter activity in both hematopoietic cells and in Sp1 null Drosophila S2 cells. In addition, Ku binds to this domain in a sequence-specific manner. Activation of the CD34 promoter by Sp1 requires the presence of a binding domain at -48 bp as well as the 5' untranslated region, which also binds Sp1.A functional interaction between regulatory regions upstream and downstream of the transcription start site is required for CD34 gene expression.
Animals↗
Translocation of Ku86/Ku70 to the multiple myeloma cell membrane: functional implications.
OBJECTIVE: Since the central hallmarks of human multiple myeloma (MM) are abnormalities in immunoglobulin (Ig) gene rearrangement, IgH class switching, and DNA damage repair, and since Ku86 and Ku70 proteins are central to these processes, aberrant Ku function may play a role in MM pathogenesis. Our prior studies demonstrated a 69-kDa Ku86 variant in freshly isolated patient MM cells that confers sensitivity to DNA damage. We also showed that Ku86 on the cell surface of CD40-activated MM cells mediates homotypic tumor cell adhesion, as well as heterotypic adhesion to bone marrow stromal cells. We here define the mechanism and functional significance of CD40-induced Ku translocation from the cytoplasm to the cell membrane in MM cells vs normal B cells. MATERIALS AND METHODS: We examined Ku86 and Ku70 translocation following CD40 activation in human MM cells vs normal tonsillar B lymphocytes. We then identified the functional sequelae of membrane Ku86 and Ku70 expression on CD40-activated human MM cells. RESULTS: CD40 activation induces translocation of both Ku86 and Ku70 to the cell surface of MM cells, but not normal tonsillar B cells. Moreover, CD40 activation triggers Ku association with CD40 only in CD40-activated MM cells. Finally, CD40-activated MM cells adhere to fibronectin and are protected against apoptosis triggered by irradiation or doxorubicin; conversely, antibodies to Ku both inhibit tumor cell binding and restore sensitivity to these agents. CONCLUSION: These results demonstrate functional significance of Ku translocation to the cell membrane of CD40-activated human MM cells. Therefore, targeting Ku86 and Ku70, with blocking peptides for example, might serve as a novel treatment strategy in human MM.
Antigens, Nuclear↗
Expression of DNA-dependent protein kinase catalytic subunit and Ku80 in developing human brains: implication of DNA-repair in neurogenesis.
We investigated expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku80 in developing human brain, both of which have been suggested to be involved in the repair of DNA double-strand break (DSB). Their expressions were well correlated, and the highest immunoreactivity was observed in post-mitotic immature neurons in the cerebral cortex as well as in progenitors in the periventricular germinal layer. The reactivity gradually decreased during development. Our results support the notion that DSB is generated during the ontogeny of the human central nervous system.
Adolescent↗
Expression of genes involved in repair of DNA double-strand breaks in normal and tumor tissues.
BACKGROUND: DNA double-strand breaks (DSB) are the major lethal lesions induced by ionizing radiation. The capability for DNA DSB repair is crucial for inherent radiosensitivity of tumor and normal cells. DNA-PKcs, Ku 70, Ku 85, Xrcc4, and Nbs1 play a critical role in DNA DSB repair. METHODS: We immunohistochemically investigated the expression of DNA-PKcs, Ku 70, Ku85, Xrcc4, and Nbs1 in 134 specimens from various normal and tumor tissues with different radiosensitivity. RESULTS AND CONCLUSION: Immunopositivity to Ku70, Ku85, DNA-PKcs, Xrcc4, and Nbs1 was found in all tumor tissues examined. The staining for Ku70, Ku85, and DNA-PKcs was nuclear; but, for Xrcc4 and Nbs1, it was nuclear and cytoplasmic. There were no apparent differences in the expression of these five proteins among cancerous tissues and the corresponding normal tissues. No apparent differences in nuclear staining intensity were detected in the expression of these five proteins among tumor tissues with different radiosensitivity, although non-Hodgkins' lymphoma (B or T cell) tended to show a lower expression than the others. The stromal cells generally expressed these five proteins at much lower frequency than either tumor or epithelial cells in both tumor and normal tissues.
Antigens, Nuclear↗
c-erbB-2 oncoprotein expression related to chemoradioresistance in esophageal squamous cell carcinoma.
PURPOSE: Esophageal carcinoma is a challenging target for radiotherapy. To improve treatment efficacy, an investigation of a predictive factor is desirable. In this study, we evaluated the significance of apoptosis and immunohistochemical staining for p53, Ki-67, c-erbB-2 (HER-2/neu), Ku (p70/p80), and DNA-PKcs for predictive markers of the responsiveness to chemoradiotherapy in esophageal squamous cell carcinoma. MATERIALS AND METHODS: This retrospective analysis consisted of 34 patients with esophageal squamous cell carcinoma in whom tumor biopsy was performed before treatment. They were divided into chemoradiosensitive (n = 13) and chemoradioresistant (n = 21) groups according to the tumor response evaluated at a total radiation dose of 40 Gy. The biopsy samples were examined with immunohistochemical staining for various factors and with an in situ nick end labeling method for apoptosis. The examined data were compared between the two groups. RESULTS: The difference in the Ki-67, p53, Ku (p70/p80), DNA-PKcs labeling indexes and the apoptosis index in tumor cells between the chemoradiosensitive and chemoradioresistant groups was not statistically significant. The expression of c-erbB-2 oncoprotein was statistically significant in the chemoradioresistant group (p = 0.02), although it did not correlate with survival. CONCLUSIONS: c-erbB-2 immunostaining is useful for the prediction of chemoradioresistance in esophageal squamous cell carcinoma.
Adult↗
The immunohistochemical expression of DNA-PKCS and Ku (p70/p80) in head and neck cancers: relationships with radiosensitivity.
PURPOSE: The DNA-PK complex is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. This study evaluated the relationship between the immunohistochemical expression of the individual components of DNA-PK and cellular radiosensitivity in head and neck cancers. METHODS AND MATERIALS: Biopsies from patients with previously untreated squamous cell carcinomas of the head and neck were assessed for inherent tumor radiosensitivity measured as the surviving fraction at 2 Gy (SF2) using a soft agar clonogenic assay. Paraffin-embedded tumor material from 64 successfully grown specimens was immunohistochemically stained for expression of DNA-PKcs and Ku (p70/p80). The same tumor material was previously analyzed for the immunohistochemical expression of p53. RESULTS: A significant correlation was found between the degree of expression of DNA-PKcs and Ku (p70/p80) (r = 0.55, p<0.001). There were no overall significant differences in the levels of expression of DNA-PKcs and Ku (p70/p80) in tumors from patients of either sex, different sites, histologies, and stages. No relationship was found between SF2 and the expression of either DNA-PKcs (r = 0.22, p = 0.081) or Ku (p70/p80) (r = 0.064, p = 0.62). Comparison with previous immunohistochemical characterization showed no significant correlations between the expression levels of p53 and either DNA-PKcs (r = 0.093, p = 0.46) or Ku (p70/p80) (r = -0.17, p = 0.17). CONCLUSIONS: This study suggests that determining the immunohistochemical expression of DNA-PK in head and neck cancers from multiple sites does not have a role as a predictive assay of tumor in vitro radiosensitivity.
Antigens, Nuclear↗