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Differential Modulation of Ku70/80 DNA-Binding Activity in a Patient with Multiple Basal Cell Carcinomas

2003; Elsevier BV; Volume: 121; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2003.12416.x

1523-1747

Paola Mazzarelli, Carla Rabitti, Paola Parrella, Davide Seripa, Paolo Persichetti, Giovanni Francesco Marangi, Giuseppe Perrone, Maria Luana Poeta, Mario Delfino, Vito Michele Fazio,

Hedgehog Signaling Pathway Studies

Ku70/80 nonhomologous end-joining activity is essential for resolving random DNA double-strand breaks, and the Ku70/80 protein complex has been proposed as "caretaker" of genomic stability. We studied the Ku70/80 heterodimer activity in a patient affected by multiple basal cell carcinomas with a personal history of moderate exposure to ionizing radiation. The Ku70/80 DNA-binding activity was analyzed, by electrophoretic mobility shift assay, in five tumor biopsies from different sites and at distinct clinical stages, and in three matched normal skin samples from the same patient. As control normal tissues from healthy individuals were also tested. The five basal cell carcinomas were classified as "non aggressive" and "aggressive" on the basis of morphologic parameters and expression of the molecular markers bcl-2, Ki67/MIB1, and p53. A 62% increase in the Ku70/80 DNA-binding activity was found in normal skin from the patient, compared to unexposed individuals (p<0.0001). The nuclear activity of the heterodimer was further increased in nonaggressive basal cell carcinomas compared to both matched normal skin from the patient (31%, p=0.0001) and tissues from healthy controls (73%, p=0.0001). Strikingly, the two aggressive basal cell carcinomas tested showed very low Ku70/80 DNA-binding activity with a reduction of 87% compared to normal skin from the patient (p<0.0001) and 64% compared to controls (p=0.001). Although these results are limited to only one patient, together with other recent studies they support the hypothesis that downregulation of the nonhomologous end-joining pathway may be associated with tumor progression. Ku70/80 nonhomologous end-joining activity is essential for resolving random DNA double-strand breaks, and the Ku70/80 protein complex has been proposed as "caretaker" of genomic stability. We studied the Ku70/80 heterodimer activity in a patient affected by multiple basal cell carcinomas with a personal history of moderate exposure to ionizing radiation. The Ku70/80 DNA-binding activity was analyzed, by electrophoretic mobility shift assay, in five tumor biopsies from different sites and at distinct clinical stages, and in three matched normal skin samples from the same patient. As control normal tissues from healthy individuals were also tested. The five basal cell carcinomas were classified as "non aggressive" and "aggressive" on the basis of morphologic parameters and expression of the molecular markers bcl-2, Ki67/MIB1, and p53. A 62% increase in the Ku70/80 DNA-binding activity was found in normal skin from the patient, compared to unexposed individuals (p<0.0001). The nuclear activity of the heterodimer was further increased in nonaggressive basal cell carcinomas compared to both matched normal skin from the patient (31%, p=0.0001) and tissues from healthy controls (73%, p=0.0001). Strikingly, the two aggressive basal cell carcinomas tested showed very low Ku70/80 DNA-binding activity with a reduction of 87% compared to normal skin from the patient (p<0.0001) and 64% compared to controls (p=0.001). Although these results are limited to only one patient, together with other recent studies they support the hypothesis that downregulation of the nonhomologous end-joining pathway may be associated with tumor progression. basal cell carcinoma DNA protein kinase catalytic subunit double-strand break nonhomologous end-joining Cancer is a multistep process characterized by the accumulation of multiple genetic alterations involving oncogenes and tumor suppressor genes, in the evolution from a normal cell to a malignant phenotype (Fearon and Vogelstein, 1990Fearon E.R. Vogelstein B. A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9466) Google Scholar). Most of the tumor suppressor genes can be divided into two categories: "gatekeeper" and "caretaker". Gatekeeper genes inhibit the cell cycle and promote cell death, whereas caretaker genes act indirectly monitoring the stability of the genome and suppressing chromosomal translocation. Inactivation of caretakers leads to genetic instability that promotes growth by causing an increased mutation rate (Kinzler and Vogelstein, 1997Kinzler K.W. Vogelstein B. Gatekeepers and caretakers.Nature. 1997; 386: 761-763Crossref PubMed Scopus (969) Google Scholar). A caretaker role has recently been proposed for the nonhomologous end-joining (NHEJ) pathway of DNA repair (Difilippantonio et al., 2000Difilippantonio M.J. Zhu J. Chen H.T. et al.A DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation.Nature. 2000; 404: 510-514Crossref PubMed Scopus (457) Google Scholar;Ferguson et al., 2000Ferguson D.O. Sekiguchi J.M. Chang S. Frank K.M. Gao Y. DePinho R.A. Alt F.W. The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.Proc Natl Acad Sci USA. 2000; 97: 6630-6633Crossref PubMed Scopus (305) Google Scholar;Gao et al., 2000Gao Y. Ferguson D.O. Xie W. et al.Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.Nature. 2000; 404: 897-900Crossref PubMed Scopus (478) Google Scholar). This mechanism is critical for resolving DNA double-strand breaks (DSB), which if left unrepaired lead to broken chromosomes and cell death, but if repaired improperly can lead to genomic instability and oncogenic rearrangement (Chu, 1997Chu G. Double strand break repair.J Biol Chem. 1997; 272: Z4097-Z4100Google Scholar;Khanna and Jackson, 2001Khanna K.K. Jackson S.P. DNA double-strand breaks: Signaling, repair and the cancer connection.Nature Gen. 2001; 27: 247-254Crossref PubMed Scopus (1798) Google Scholar). This mechanism of DNA repair is mediated by the DNA-binding activity of the Ku70/80 heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) (Jin and Weaver, 1997Jin S. Weaver D. Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.EMBO J. 1997; 16: 6874-6885Crossref PubMed Scopus (122) Google Scholar). In murine models, cells deficient for Ku80 or Ku70 display a marked increase in chromosomal aberrations (Difilippantonio et al., 2000Difilippantonio M.J. Zhu J. Chen H.T. et al.A DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation.Nature. 2000; 404: 510-514Crossref PubMed Scopus (457) Google Scholar;Roth and Gellert, 2000Roth D.B. Gellert M. New guardians of the genome.Nature. 2000; 404: 823-825Crossref PubMed Scopus (37) Google Scholar). Moreover genomic instability and development of B cell lymphoma were reported in two lines of mice lacking both the NHEJ protein Ku80 and the p53 gene, supporting the hypothesis of a possible role as caretaker for the NHEJ proteins (Lim et al., 2000Lim D.S. Vogel H. Willerford D.M. Sands A.T. Platt K.A. Hasty P. Analysis of Ku80-mutant mice and cells with deficient levels of p53.Mol Cell Biol. 2000; 20: 3772-3780Crossref PubMed Scopus (135) Google Scholar;Roth and Gellert, 2000Roth D.B. Gellert M. New guardians of the genome.Nature. 2000; 404: 823-825Crossref PubMed Scopus (37) Google Scholar). DSB can be caused by both endogenous and exogenous agents. Oxidative metabolism generates free radicals that may cause strand breaks and DSB are also generated as a consequence of the V(D)J recombination, during the rearrangement of genes for the immunoglobulins and T cell receptors (Smider and Chu, 1997Smider V. Chu G. The end-joining reaction in V(D)J recombination.Semin Immunol. 1997; 9: 189-197Crossref PubMed Scopus (52) Google Scholar), whereas the main exogenous mechanism responsible for DSB is exposure to ionizing radiation (Chu, 1997Chu G. Double strand break repair.J Biol Chem. 1997; 272: Z4097-Z4100Google Scholar). Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are among the tumors associated with exposure to ionizing radiation (Karagas et al., 1996Karagas M.R. McDonald J.A. Greenberg R. Stukel T.A. Weiss J.E. Baron J.A. Stevens M.M. Risk of basal cell and squamous cell skin cancers after ionizing radiation therapy.J Natl Cancer Inst. 1996; 88: 1848-1853Crossref PubMed Scopus (175) Google Scholar;Lichter et al., 2000Lichter M.D. Karagas M.R. Mott L.A. Spencer S.K. Stukel T.A. Greenberg E.R. Therapeutic ionizing radiation and the incidence of basal cell carcinoma and squamous cell carcinoma.Arch Dermatol. 2000; 136: 1007-1011Crossref PubMed Scopus (148) Google Scholar). The risk of BCC and SCC development is strongly increased after ionizing radiation therapies or X-ray exposure (Karagas et al., 1996Karagas M.R. McDonald J.A. Greenberg R. Stukel T.A. Weiss J.E. Baron J.A. Stevens M.M. Risk of basal cell and squamous cell skin cancers after ionizing radiation therapy.J Natl Cancer Inst. 1996; 88: 1848-1853Crossref PubMed Scopus (175) Google Scholar;Lichter et al., 2000Lichter M.D. Karagas M.R. Mott L.A. Spencer S.K. Stukel T.A. Greenberg E.R. Therapeutic ionizing radiation and the incidence of basal cell carcinoma and squamous cell carcinoma.Arch Dermatol. 2000; 136: 1007-1011Crossref PubMed Scopus (148) Google Scholar), and these malignancies have been described in individuals exposed to the atomic bombing of Hiroshima and Nagasaki (Ron et al., 1998Ron E. Preston D.L. Kishikawa M. et al.Skin tumor risk among atomic-bomb survivors in Japan.Cancer Causes Control. 1998; 9: 393-401Crossref PubMed Scopus (117) Google Scholar). However, the relationship between DNA DSB repair and risk for skin cancer is not fully understood (Grossman and Wie, 1995Grossman L. Wie Q. DNA repair and epidemiology of basal cell carcinoma.Clin Chem. 1995; 41: 1854-1863PubMed Google Scholar; Gottlober et al., 1999Gottlober P. Krahn G. Bezold G. Peter R.U. Basal cell carcinomas occurring after accidental exposure to ionizing radiation.Br J Dermatol. 1999; 141: 383-385Crossref PubMed Scopus (11) Google Scholar). In this study, we have analyzed the Ku70/80 DNA-binding activity in skin tumor biopsies and matched normal skin from a patient affected by multiple BCC and with a personal history of moderate exposure to ionizing radiation. As control, normal tissues from healthy individuals were also tested. We found a striking correlation between Ku70/80 DNA-binding activity and tumor progression. The study was carefully examined by the Ethical Committee of the Campus Bio-Medico University. The patient is a 75-y-old white male, skin type III (Fitzpatrick classification) (De Vita et al, 1997), who developed multiple BCC, a single SCC, and aplastic anemia during the period from 1986 to 1999. The patient worked as a radiologist for 6 y, from 1955 to 1961. In 1986 he lived in Budapest from the week preceding until 2 wk following the Chernobyl accident. No data are available about the radiation exposure rate for our patient. The 1988 Report of the United Nations Scientific Committee on the Effects of Atomic Radiations (UNSCEAR) (UNSCEAR Report, 1988UNSCEAR Report Annex D: Exposures from the Chernobyl Accident. UNSCEAR. 1988: 1-74Google Scholar), however, indicates in the region of Budapest an outdoor effective dose equivalent in the first month from external irradiation of 100 μSv. The patient developed the first BCC on the forehead and then another 11 BCC were diagnosed. He was last seen in our clinical facility in 1999. The SCC and nine of the 12 BCC were excised. The patient denies previous neoplastic pathologies and radiation therapy. Absence of palmar pits, mandibular cysts, calcification of the falx cerebri, bifid ribs, and a negative family history exclude the nevoid BCC syndrome (Gailani and Bale, 1997Gailani M.R. Bale A.E. Developmental genes and cancer: Role of patched in basal cell carcinoma of the skin.J Natl Cancer Inst. 1997; 89: 1103-1109Crossref PubMed Scopus (96) Google Scholar). Five BCC and two matched normal skin biopsies (Table I), surgically excised at the Plastic Surgery Division of Campus Bio-Medico University, were analyzed in this study. As controls, five normal skin sections from healthy subjects together with one normal sample from human bladder and three breast tissues were also tested. Informed consent was obtained from all the subjects included in the study. All specimens underwent histologic examination to confirm the diagnosis. Tumor samples were classified morphologically on the basis of growth pattern, as described previously (Jacobs et al., 1982Jacobs G.H. Rippey J.J. Altini M. Prediction of aggressive behavior in basal cell carcinoma.Cancer. 1982; 49: 533-537Crossref PubMed Scopus (198) Google Scholar;Rippey and Rippey, 1997Rippey J.J. Rippey E. Characteristics of incompletely excised basal cell carcinomas of the skin.Med J Aust. 1997; 166: 581-583PubMed Google Scholar).Table IClinical characteristics and immunohistochemical results of multiple BCCs (T1–T5) from the same patientCodeHistologic subtypeaBBCs were discriminated by histological growth patterns according to Rippey and colleagues (Rippey et al, 1998).SiteOnsetSize (cm)p53 (%)bPercent values of positively stained cells.Ki-67bcl-2cn.d., not detected.T1NodularForehead0.56228n.d.T2NodularLeft legPrimary0.55527+T3SuperficialLeft leg0.85731+++T4InfiltrativeRight legRecurrent1.057464NegT5MicronodularBackPrimary19159Nega BBCs were discriminated by histological growth patterns according to Rippey and colleagues (Rippey et al, 1998).b Percent values of positively stained cells.c n.d., not detected. Open table in a new tab To exclude that the patient was affected by nevoid BCC syndrome, DNA extracted from fresh frozen tumor samples and blood lymphocytes was analyzed for loss of heterozygosity in microsatellite markers mapping on chromosome 9q near the nevoid BCC syndrome locus (Wicking et al., 1994Wicking C. Berkman J. Wainwright B. Chenevix-Trench G. Fine genetic mapping of the gene for nevoid basal cell carcinoma syndrome.Genomics. 1994; 22: 505-511Crossref PubMed Scopus (46) Google Scholar). The following markers were tested: D9S196 (9q22); D9S197 (9q22.1); D9S180, D9S12, D9S287 (9q22,3); D9S109, D9S127 (9q31) (Farndon et al., 1992Farndon P.A. Del Mastro R.G. Evans D.G. Kilpatrick M.W. Location of gene for Gorlin syndrome.Lancet. 1992; 339: 581-582Abstract PubMed Scopus (315) Google Scholar;Goldstein et al., 1994Goldstein A.M. Stewart C. Bale A.E. Bale S.J. Dean M. Localization of the gene for the nevoid basal cell carcinoma syndrome.Am J Hum Genet. 1994; 54: 765-773PubMed Google Scholar;Wicking et al., 1994Wicking C. Berkman J. Wainwright B. Chenevix-Trench G. Fine genetic mapping of the gene for nevoid basal cell carcinoma syndrome.Genomics. 1994; 22: 505-511Crossref PubMed Scopus (46) Google Scholar). PCR conditions included a denaturation step for 2 min at 95°C, followed by 35 cycles at 95°C for 1 min, 54–58°C for 1 min, 72°C for 1 min, and by a final 4 min extension at 72°C (Seripa et al., 2001Seripa D. Parrella P. Gallucci M. et al.Sensitive detection of transitional cell carcinoma of the bladder by microsatellite analysis of cells exfoliated in urine.Int J Cancer. 2001; 93: 364-369Crossref Scopus (56) Google Scholar). About one-quarter of the PCR product was separated on a 7% urea formamide polyacrylamide gel and exposed to film (Parrella et al., 2001Parrella P. Xiao Y. Fliss M. et al.Detection of mitochondrial DNA mutations in primary breast cancer and fine needle aspirates.Cancer Res. 2001; 61: 7623-7626PubMed Google Scholar). Following surgical resection, tissue samples were immediately processed for protein extraction, according to the Dignam method (Dignam et al., 1983Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9046) Google Scholar]) previously described byPucci et al., 2001Pucci S. Mazzarelli P. Rabitti C. et al.Tumor specific modulation of Ku70/80 DNA binding activity in breast and bladder human tumor biopsies.Oncogene. 2001; 20: 739-747Crossref PubMed Scopus (78) Google Scholar. Briefly, samples (0.1–0.5 g in weight) were put on ice and mechanically fractionated to obtain a cellular suspension. Red blood cells were depleted by osmotic lysis. Cells were then washed twice and suspended in 0.5 ml of ice-cold buffer A (HEPES-KOH 10 mM, pH 7.9, MgCl2 1.5 mM, KCl 10 mM, ethylenediamine tetraacetic acid (EDTA) 1 mM, and dithiothreitol (DTT) 1 mM, phenyl-methylsulfonyl fluoride (PMSF) 1 mM, NaF 20 mM, Na4P2O7 1 mM added just prior to use); cells were allowed to swell on ice for 20 min and then 25 μL of Nonidet P-40 10% (Fluka-Sigma, St Louis, MO) was added. The homogenate was centrifuged for 30 s in a microfuge at 5000oxg (the postnuclear extract containing cytoplasmic proteins was carefully removed and stored at -80°C). The nuclear pellet was resuspended in ice-cold NaCl extraction buffer (HEPES-KOH 20 mM, pH 7.9, NaCl 420 mM, MgCl2 1.5 mM, EDTA 1 mM, glycerol 25%, and DTT 1 mM, PMSF 1 mM, NaF 20 mM, Na4P2O7 1 mM added just prior to use) and incubated on ice for 30 min. Cellular debris was removed by centrifugation for 5 min in a microfuge at 10,000 rpm.The supernatant fraction, containing DNA-binding proteins, was stored at –80°C. All steps of protein extraction were checked by optical microscope. Protein content in the nuclear extracts was determined in triplicate by Bradford assay (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munchen, Germany). All the nuclear extracts were analyzed by electrophoretic mobility shift assay (Zhang and Yaneva, 1992Zhang W.W. Yaneva M. On the mechanism of Ku protein binding to DNA.Biochem Biophys Res Commun. 1992; 186: 574-579Crossref PubMed Scopus (74) Google Scholar). A 32P-end labeled 56 bp DNA probe (Frasca et al., 1998Frasca D. Barattini P. Goso C. et al.Cell proliferation and Ku protein expression in ageing humans.Mech Ageing Dev. 1998; 100: 197-208Crossref PubMed Scopus (14) Google Scholar) was incubated with nuclear extracts for 30 min at room temperature in binding buffer (10 mM Tris-HCl pH 8, EDTA 0.5 mM, NaCl 150 mM, DTT 1 mM, PMSF 1 mM, glycerol 10%); briefly, the DNA-binding reactions contained the labeled probe (50,000 cpm), nuclear (2 μg) extracts, and closed circular plasmid DNA pUC-19 (1 μg), as unspecific competitor. For each sample three single shift assays were performed. To normalize all the samples, an electrophoretic mobility shift assay was performed incubating the nuclear extracts (2 μg) with 50,000 cpm per sample of 32P-end labeled Sp-1 oligonucleotide (Promega, Madison, WI) in binding buffer (glycerol 20%, MgCl2 5 mM, EDTA 2.5 mM, NaCl 250 mM, Tris-HCl pH 7.5 50 mM, DTT 2.5 mM) with 1 μg of poly[dI-dC] as unspecific competitor. The correction factor (CF) was calculated as follows: Sp-1 binding activity in the samplemean Sp-1 binding activityand data were normalized using the formulaMean Ku70/80 binding activityCF For gel supershift experiments goat polyclonal anti-Ku70 and anti-Ku86 antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) were incubated with protein extracts for 30 min at room temperature, before adding the other components of the binding reaction. Complexes were separated on 6% nondenaturing polyacrylamide gels, in TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) at 200 V for 2 h 30 min.Gels were dried and exposed to X-ray film (Amersham-Pharmacia Biotech, Buckinghamshire, UK) overnight at -80°C. The optical densities were obtained by scanning densitometry using colon carcinoma cell line CaCo2 (ATCC) as internal control (optical density=5.41±0.71). Protein extracts (10 μg) were boiled in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and separated by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride membrane (Hybond-P, Amersham-Pharmacia Biotech) using an electroblotting apparatus and incubated for 1 h at room temperature with 1% bovine serum albumin (BSA), 1% skim milk (Difco Laboratory, Detroit, MI), and 0.5% Tween-20 (USB, Cleveland, OH). Membranes were stained with Poinceau S dye, to check for equal loading and homogeneous transfer. Primary antibodies (goat polyclonal IgG anti-Ku70 or Ku86, Santa Cruz Biotechnologies) were diluted 1:500 in 1% BSA and incubated for 1 h at room temperature; samples were washed extensively with 0.5% Tween-20, and diluted 1:8000 secondary antibody (antigoat horseradish peroxidase conjugated IgG, Santa Cruz Biotechnologies) was added in 1% BSA, 1% milk, 0.5% Tween-20, for 1 h at room temperature. Filters were reprobed with anti-β-actin (Sigma-Aldrich, St Louis, MO) mouse IgG1 monoclonal antibody, to normalize the nuclear protein levels. Filters were washed and developed using an enhanced chemiluminescence system (ECL, Amersham-Pharmacia Biotech, Pharmacia Biotech). The specimens were fixed in 10% neutral-buffered formaldehyde and embedded in paraffin. Based on an initial review of all available hematoxylin and eosin stained slides of the surgical specimen sections, we selected one representative paraffin block from each case for further study. Consecutive 3 μm sections were re-cut from each study block; these sections were immunoassayed for p53, MIB1 (Ki67), bcl-2, Ku86, Ku70, and DNA protein kinase catalytic subunit (DNA-PKcs). Immunohistochemical staining was performed by the streptavidin-biotin method. In brief, sections were de-paraffinized and microwave-treated at 500 W for 5 min twice in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase in the sections was blocked by incubating them in 0.03% hydrogen peroxide in absolute methanol for 30 min at room temperature. The antibodies used were monoclonal mouse antibodies against human p53 protein (D07, Dakopatts, Glastrup, Denmark) in a 1:50 dilution, human bcl-2 protein (Dakopatts) in a 1:40 dilution, and anti-Ki67 protein (MIB1 clone, Dakopatts) in a 1:50 dilution; and goat polyclonal rabbit antibodies against Ku86 (M-20, Santa Cruz Biotechnology) and Ku70 proteins (M-19, Santa Cruz Biotechnology) in a 1:200 dilution and anti-DNA-PKcs protein (C-19, Santa Cruz Biotechnology) in a 1:100 dilution. All primary antibodies were incubated at room temperature for 2 h. After washing three times with Tris-buffered saline (TBS), sections treated with anti-p53, bcl-2, and Ki67 were incubated with biotinylated goat antimouse/rabbit IgG (Dako) and sections treated with anti-Ku70, Ku86, and DNA-PKcs were incubated with biotinylated swine antigoat/mouse/rabbit IgG (Dako). They were then washed three times with TBS, treated with streptavidin peroxidase reagent (Dakopatts) for 10 min, and washed with TBS three times again. Finally, specimens were incubated in diaminobenzidine for 5 min, followed by hematoxylin counterstaining. Sections were examined under a two-head microscope by two pathologists unaware of the clinical data and molecular results. All values provided in the text and figures are means of three independent experiments ±standard deviations (SD). Mean values were compared using the two-tailed t test. Differences were considered statistically significant for p 0.1). Normal skin samples from the patient showed a 62% increase in the Ku70/80 DNA-binding activity compared to tissues from healthy controls (p<0.0001) (Table III). In the three no aggressive BCC a further increase in the heterodimer binding activity could be demonstrated compared to tissues from healthy controls (p=0.0001) and matched normal skin from the patient (p=0.001) (Table III), whereas the two aggressive BCC showed a dramatic decrease in the Ku70/80 DNA-binding activity with a reduction of 64% compared to controls (p=0.001) and 87% compared to normal skin from the patient (p<0.0001) (Table III).Table IIIKu70/80 DNA-binding activity and protein expression in control skin specimens (C1–C5), normal skin (Sk1–Sk2) and BCCs (T1–T5) from the skin cancer patientCodeHistologic subtypeKu70 (%)aPercent values of positively stained cells.Ku86 (%)aPercent values of positively stained cells.DNA-PKcs (%)aPercent values of positively stained cells.Ku70/80 DNA-binding activity (O.D. means±SD)bOptical densities obtained from the densitometric analysis of the bands corresponding to the specific DNA-binding activity, in mobility-shift assays; values are means±standard deviations of three independent experiments.C1–C5Normal skin6242451.4±0.4—Sk1Normal skin7061603.7±0.4(P<0.0001)cPatient normal skin vs C1–C5 (Student's T-test).Sk2725562T1No aggressive7354595.3±0.5(P<0.0001)dTumors vs C1–C5 (Student's T-test).T2726166(P=0.001)eTumors vs Sk1–Sk2 (Student's T-test).T3687085T4Aggressive9763620.5±0.4(P<0.001)dTumors vs C1–C5 (Student's T-test).T5956269(P<0.0001)eTumors vs Sk1–Sk2 (Student's T-test).a Percent values of positively stained cells.b Optical densities obtained from the densitometric analysis of the bands corresponding to the specific DNA-binding activity, in mobility-shift assays; values are means±standard deviations of three independent experiments.c Patient normal skin vs C1–C5 (Student's T-test).d Tumors vs C1–C5 (Student's T-test).e Tumors vs Sk1–Sk2 (Student's T-test). Open table in a new tab To determine whether differences in DNA-binding activity were due to changes in protein expression, Ku70, Ku86, and DNA-PKcs protein levels were determined by immunohistochemical assay (Table III, Figure 3) and western blot analysis in nuclear extracts from patient tissues (Figure 2c). Surprisingly, no significant changes in protein expression were detected in the two tumors with decreased Ku70/80 DNA-binding activity tested, suggesting that proteins are expressed but not functional (Table III, Figure 2c, Figure 3). Upregulation of the Ku70 and Ku80 subunits following ionizing radiation exposure has been reported previously (Featherstone and Jackson, 1999Featherstone C. Jackson S.P. Ku, a DNA repair protein with multiple cellular functions?.Mutat Res. 1999; 434: 3-15Crossref PubMed Scopus (230) Google Scholar;Brown et al., 2000Brown K.D. Lataxes T.A. Shangary S. Mannino J.L. Giardina J.F. Chen J. Baskaran R. Ionizing radiation exposure results in up-regulation of Ku70 via a p53/ataxia-telangiectasia-mutated protein-dependent mechanism.J Biol Chem. 2000; 275: 6651-6656Crossref PubMed Scopus (56) Google Scholar). In this study we tested the Ku70/80 DNA-binding activity and protein expression in a patient who developed multiple BCC, and with a personal history of moderate radiation exposure. As control, the heterodimer binding activity was also determined on tissues from healthy controls. The first interesting point is the increase in the Ku70/80 DNA-binding activity in normal skin from the patient compared to healthy controls, suggesting a baseline activation of the NHEJ DNA repair pathway in the skin of this individual. When the Ku70/80 DNA-binding activity was tested on the five BCC we found two different behaviors. In three of the tumors the heterodimer DNA-binding activity was increased compared to matched skin and controls, whereas the remaining two tumors displayed a dramatic decrease of the activity. We correlated our results with the morphologic and molecular characteristics of the five tumors. BCC generally show a favorable clinical behavior, but a percentage of them grow aggressively, infiltrating contiguous structures. A number of authors have adopted a histopathologic classification of BCC based on tumor growth patterns that identify four possible classes: nodular, infiltrative, superficial, and mixed (Rippey, 1998Rippey J.J. Why classify basal cell carcinomas?.Histopathology. 1998; 32: 393-398Crossref PubMed Scopus (179) Google Scholar;Milroy et al., 2000Milroy C.J. Horlock N. Wilson G.D. Sanders R. Aggressive basal cell carcinoma in young patients: Fact or fiction?.Br J Plast Surg. 2000; 53: 393-596Abstract Full Text PDF PubMed Scopus (26) Google Scholar). Infiltrative and micronodular tumors are more likely to be incompletely excised; thus they recur more frequently and are considered as aggressive. A role as prognostic factor in BCC has been proposed for bcl-2 and p53. bcl-2 expression was directly correlated with nonaggressive BCC and a favorable clinical follow-up, whereas the expression of p53 was correlated with the aggressive histotypes (Barrett et al., 1997Barrett T.L. Smith K.J. Hodge J.J. Butler R. Hall F.W. Skelton H.G. Immunohistochemical nuclear staining for p53, PCNA, and Ki-67 in different histologic variants of basal cell carcinoma.J Am Acad Dermatol. 1997; 37: 430-437Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar;Ramdial et al., 2000Ramdial P.K. Madaree A. Reddy R. Chetty R. Bcl-2 protein expression in aggressive and non-aggressive basal cell carcinomas.J Cutan Pathol. 2000; 27: 283-291Crossref PubMed Scopus (49) Google Scholar;Staibano et al., 2001Staibano S. Lo Muzio L. Pannone G. et al.Interaction between bcl-2 and p53 in neoplastic progression of basal cell carcinoma of the head and neck.Anticancer Res. 2001; 21: 3757-3764PubMed Google Scholar). Moreover the expression of the proliferation marker Ki67/MIB1 also directly correlates with aggressiveness (Healy et al., 1995Healy E. Angus B. Lawrence C.M. Rees J.L. Prognostic value of Ki67 antigen expression in basal cell carcinomas.Br J Dermatol. 1995; 133: 737-741Crossref PubMed Scopus (65) Google Scholar). Strikingly, the two tumors with a decrease in Ku70/80 binding activity showed all the characteristics of aggressive tumors, whereas the three BCC with high heterodimer binding activity displayed the characteristics of a no aggressive phenotype (Table I). Our data on normal skin and nonaggressive BCC seem to support the hypothesis that the increase of Ku70/80 DNA-binding activity represents a physiologic mechanism, attempting to prevent or reduce the development of genomic instability. It is more difficult to explain the decrease in heterodimer binding activity in the aggressive BCC. This might be due to post-translational regulation, or alternatively to mutation or allele inactivation in one or more of the elements involved in the NHEJ pathway. Unfortunately we did not have enough material to perform mutational and expression analysis on the Ku70 and Ku80 genes to further investigate this issue. In both cases, however, we can speculate that the failure of the NHEJ pathway may be responsible for genetic instability and lead ultimately to tumor progression. In animal models the inactivation of the NHEJ pathway increases the tumorigenic effect determined by loss of function of the p53 protein (Difilippantonio et al., 2000Difilippantonio M.J. Zhu J. Chen H.T. et al.A DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation.Nature. 2000; 404: 510-514Crossref PubMed Scopus (457) Google Scholar;Gao et al., 2000Gao Y. Ferguson D.O. Xie W. et al.Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.Nature. 2000; 404: 897-900Crossref PubMed Scopus (478) Google Scholar;Khanna and Jackson, 2001Khanna K.K. Jackson S.P. DNA double-strand breaks: Signaling, repair and the cancer connection.Nature Gen. 2001; 27: 247-254Crossref PubMed Scopus (1798) Google Scholar). Interestingly, in our patient p53 is overexpressed in all five BCC, but only in the two aggressive tumors did we also find an inactivation of the NHEJ pathway. In fact, in physiologic conditions the p53 protein is rapidly degraded through interaction with the MDM2 gene product and it is not detected by immunohistochemical staining. p53 overexpression occurs in the presence of mutations that cause disruption of the p53–MDM2 interaction leading to elevated levels of an inactive p53 protein (Umekita et al., 1994Umekita Y. Kobayashi K. Saheki T. Yoshida H. Nuclear accumulation of p53 protein correlates with mutations in the p53 gene on archival paraffin-embedded tissues of human breast cancer.Jpn J Cancer Res. 1994; 85: 825-830Crossref PubMed Scopus (35) Google Scholar;Prives and Hall, 1999Prives C. Hall P.A. The p53 pathway.J Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1204) Google Scholar). It remains to be determined whether the differential modulation of the NHEJ pathway is a common event in aggressive and nonaggressive tumors. Previous studies reported a downregulation of the Ku70/80 NHEJ components in aggressive and metastatic malignancies compared to benign lesions or less aggressive tumors of the same types (Pucci et al., 2001Pucci S. Mazzarelli P. Rabitti C. et al.Tumor specific modulation of Ku70/80 DNA binding activity in breast and bladder human tumor biopsies.Oncogene. 2001; 20: 739-747Crossref PubMed Scopus (78) Google Scholar;Rigas et al., 2001Rigas B. Borgo S. Elhosseiny A. et al.Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis.Cancer Res. 2001; 61: 8381-8384PubMed Google Scholar). If further studies confirm the differential modulation of the Ku70/80-DNA-PKcs complex during tumor progression, the role of the NHEJ pathway as caretaker would be proven (Ferguson et al., 2000Ferguson D.O. Sekiguchi J.M. Chang S. Frank K.M. Gao Y. DePinho R.A. Alt F.W. The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.Proc Natl Acad Sci USA. 2000; 97: 6630-6633Crossref PubMed Scopus (305) Google Scholar;Gao et al., 2000Gao Y. Ferguson D.O. Xie W. et al.Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.Nature. 2000; 404: 897-900Crossref PubMed Scopus (478) Google Scholar) and we would have a new and potentially powerful marker of prognosis. This work is dedicated to Professor Torsoli, MD, for his lifelong and passionate dedication to clinical practice, science, and medical education. We are also grateful to Professor Torsoli for kindly making available the patient's specimens and clinical information, and for illuminated scientific suggestions up to the end of his life. Funding was provided by Italian Ministero della Salute, IRCCS RC 2000 and 2001, and from IRCCS H. "Casa Sollievo Sofferenza", "Fondazione Opera di Padre Pio da Pietrelcina". P. Mazzarelli, MD, is a PhD student, Program in Experimental Dermatology, Università"Federico II", Napoli, Italy.