Genomic DNA is captured and amplified during double-strand break (DSB) repair in human cells

Genomic stability is maintained by the surveillance and repair of DNA damage. Here, we describe a mechanism whereby repair of extrachromosomal DNA double-strand breaks (DSBs) in human cells can be accompanied by capture of genomic DNA fragments. The availability of the human genome sequence enabled us to characterize these inserts in cells from a normal individual and from a patient with ataxia telangiectasia (AT), deficient for the damage response kinase ATM and prone to genomic instability. We find AT cells exhibit insertions of human chromosomal DNA fragments in excess of 17 kb during DSB repair, whereas we detected no such genomic inserts in normal cells. However, the presence of simian virus 40 (SV40), used to transform these cell lines, resulted in capture of genomic DNA associated with sites of viral integration in both cell types. The genomic instability at sites of SV40 integration was exported to other sites of DNA damage, and acquisition of the viral origin of replication resulted in gene amplification through autonomous replication of the plasmid harbouring the repaired extrachromosomal DSB. Should this same phenomenon apply to the repair of chromosomal DSBs, genome rearrangements made possible via this DSB insertional repair pose risks to genomic integrity, and may contribute to tumorigenic progression.     

Hypersensitivity of cultured ataxia-telangiectasia cells to etoposide

Fibroblasts from patients with ataxia-telangiectasia (A-T) were found to be hypersensitive to killing by the antineoplastic agent etoposide. The A-T fibroblast strains GM5823, GM367, and GM2052 were twofold to threefold more sensitive to killing by etoposide than fibroblasts from normal controls (AG1521, AG1522, and IMR90). A simian virus 40 (SV40)-transformed, immortal human fibroblast line (GM5849) derived from the A-T cell line GM5823 was also studied. GM5849 retained the unusual sensitivity of nontransformed A-T fibroblast lines to x-irradiation, bleomycin, and neocarzinostatin (zinostatin). GM5849 was also more sensitive to etoposide than were SV40-transformed fibroblasts from normal controls. M1, and SV40-transformed fibroblast line derived from a patient with xeroderma pigmentosum, had the same sensitivity to etoposide as SV40-transformed fibroblasts derived from normal controls.     

Hypersensitivity to cell killing and faulty repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in human (ataxia-telangiectasia) fibroblasts treated with 4-nitroquinoline 1-oxide

Dermal fibroblast strains from ataxia-telangiectasia (A-T) patients and clinically normal subjects were exposed to 4-nitroquinoline 1-oxide (4NQO) or its 3-methyl derivative (3me4NQO), and their colony-forming abilities and DNA metabolic properties were compared. Three A-T strains, i.e., AT2BE and AT3BI representing genetic complementation group AB and AT4BI belonging to group C, displayed enhanced (2.4- to 2.8-fold) sensitivity to reproductive inactivation by 4NQO, but exhibited normal survival in response to 3me4NQO. The initial induction and subsequent enzymatic repair of alkali-labile lesions (i.e., damaged sites converted to single-strand breaks in alkali) were quantitated by conventional velocity sedimentation analysis of cellular DNA in alkaline sucrose gradients. On exposure to concentrations of each chemical that produced comparable amounts of DNA damage, A-T and normal cells removed alkali-labile lesions at similar rates. However, the three 4NQO-sensitive A-T strains appeared to be defective in acting on alkali-stable adducts (formed by the parent compound but not its derivative), as judged by strand-break accumulation during posttreatment incubation with 1-beta-D-arabinofuranosylcytosine (araC). Specifically, the number of araC-detectable sites repaired in these A-T strains during the critical 2-h period immediately following 4NQO treatment ranged from 40 to 60% of that processed by normal controls. AT5BI, a fourth A-T strain assigned to complementation group D, responded normally to 4NQO-induced cytotoxicity and removed both alkali-labile and alkali-stable (araC-detectable) lesions normally. We thus conclude that the hypersensitivity of AT2BE, AT3BI, and AT4BI strains to 4NQO may be attributed, at least in part, to faulty execution of the excision-repair process operative on alkali-stable 4NQO adducts.     

Cellular consequences of overproduction of DNA topoisomerase II in an ataxia-telangiectasia cell line

Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs [e.g., the DNA intercalator amsacrine (mAMSA)]. To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5-10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions). Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells.     

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.     

Chromosomal imbalances and DNA amplifications in SV40 large T antigen-induced primitive neuroectodermal tumor cell lines of the rat.

Comparative genomic in situ hybridization analysis of four cell lines derived from SV40 large T antigen-induced primitive neuroectodermal tumors of the rat revealed non-recurrent chromosomal copy number changes and DNA amplifications at chromosomal bands 2q34, 4q43qter and 15q12qter in cell lines TZ102, TZ103 and TZ107, respectively. Semi-quantitative PCR and western blot analysis demonstrated amplification and over-expression of the rat N-ras proto-oncogene in TZ102. Furthermore, all cell lines displayed aneuploid cell populations and variable chromosome numbers as assessed by flow cytometry and cytogenetics. These findings suggest that DNA amplification as well as genomic instability may contribute to the pathogenesis of SV40 large T antigen-induced primitive neuroectodermal tumors of the rat.     

Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants

In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G(1) phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.     

Alterations in SV40 DNA integration patterns are associated with acquisition of the invasive phenotype in hamster brain tumors

A panel of cell lines that represents a reproducible, easily manipulated experimental system which discriminates between the minimally and diffusely invasive phenotypes of brain tumors has been developed. A population of SV40-transformed glial cells derived from newborn hamster cerebral cortex (Cx) has been sequentially passaged in newborn hamsters by intracerebral inoculation followed by in vitro culture, and after each passage progressively more invasive cell lines have been established. To study the molecular basis for the observed phenotypic characteristics associated with invasiveness, cloned cells were isolated from the first (Cx4T1-derived) and third Passage (Cx4T3-derived) cells lines. After injection into hamster brain, these cloned cells produce tumors that were either minimally invasive (Cx4T1-derived) or diffusely invasive (Cx4T3-derived) into normal brain tissue. In our initial attempt to identify and characterize the cellular and molecular factors that modulate the invasive phenotype, restriction endonuclease generated SV40 DNA-containing fragment patterns of DNA from each parental cell line and each of the clonal variants were determined by Southern transfer-hybridization. The results suggest the cell lines are composed of a limited number of tumorigenic subpopulations, each of which contain characteristic arrangements of integrated SV40 DNA with repeated in vivo/in vitro passage the avvangement of intecyvated SV40 changed. Analysis of DNA from minimally and diffusely invasive cloned cells indicated strong similarities of integrated SV40 DNA arrangement to their parental cells with the greatest similarities in cells exhibiting comparable invasive phenotypes. A striking difference was seen, however, in comparisons of SV40 DNA-containing fragment patterns of DNA extracted from clones which induced marginally versus diffusely invasive tumors. These differences suggest that the invasive cells were selected from a distinct minority subpopulation or that they may have arisen as a consequence of a more dynamic process of genetic rearrangement. This cell system appears to mimic the phenotypic and genetic heterogeneity observed in human tumors of glial origin and should prove valuable in defining the biochemical and molecular basis of tumor cell invasion.     

Increased uracil-DNA glycosylase, AP-DNA binding protein and deoxyribonuclease activities in tumor and SV40-transformed cell lines of human origin.

The activities of three human DNA metabolizing enzymes--uracil-DNA glycosylase, apurinic/apyrimidinic(AP)-DNA binding protein (an AP-DNA endonuclease) and the major cellular deoxyribonuclease (presumably DNase III and/or DNase IV)--were measured in logarithmic growing (diploid non-established) fibroblast strains, tumor-derived cell lines and SV40-transformed cell lines. The levels of activity of uracil-DNA glycosylase and DNase were increased, on average, 5- to 6-fold in tumor cell lines and 10-fold in SV40-transformed cell lines compared to those observed in normal fibroblast strains. AP-DNA binding activity was only 2- to 3-fold higher in both tumor-derived and SV40-transformed cell lines. Measurements in serum-deprived (and hence growth-retarded) SV40-transformed cells indicated that the observed increase in enzyme activity was only partially due to a higher proportion of S-phase cells in the rapidly growing transformed lines. Cell extract mixing experiments indicated that the relatively low levels of activity of the three enzymes in normal fibroblasts could not be ascribed to the presence of an inhibitory factor(s) in the crude extract.     

SV40 transfection of human kidney epithelial cells and stability of chromosome 11

Simian virus 40 (SV40) transformation has been used in a variety of mammalian cells and has been shown to extend their life span. We therefore decided to apply these results to normal kidney and tumoral cells derived from Wilms' patients. Wilms' tumour, a nephroblastoma which presents in early childhood, has been linked to deletions and rearrangements in chromosome 11. Analysis of gene structure in the 11p13 locus involved in the development of the tumour has been restricted by the very short life-span of the tumoral cells in vitro. We transfected normal kidney WT/NK, tumoral WT/T cells and human foetal kidney HFK cells with 2 SV40-derived plasmids SV3neo (pBR322-SV40-containing neomycin bacterial gene) and SVori- (pMK-origin mutated SV40). We isolated a high number of SV40-transfected cell lines. The efficiency of transfection appeared to be extremely low in WT/T cells compared with HFK and even WT/NK. The life span of the cell lines was increased in relation to their untransfected homologues. However, in all of the cell lines except 3, senescence occurred, after crisis step or not. We looked at different markers associated with SV40 transformation of mammalian cells and specifically with the presence of SV40 T antigen in the cells and its consequences: AIG, tumorigenicity, expression and insertion in genomic DNA of SV40 T antigen. Genetic studies involving karyotypic and restriction fragment length polymorphism (RFLP) analysis demonstrated that, despite a frequent pseudo-diploidy, the cell lines derived have conserved the 11p13 locus.     

DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection

During selection for methotrexate resistance, SV40-transformedhuman skin fibroblasts from patients with ataxia telangiectasia(A-T) underwent amplification of the dihydrofolate reductase(DHFR) gene, experienced nearly complete loss of the integratedSV40 sequences and showed a 3.6-fold increase in Ki-ras genecopy number. Over a period of months methotrexate-resistant(MTX^r) A-T subclones were obtained, which were able to growin progressively increasing MTX concentrations up to 100 µM.The ED₅₀ values determined as the effective dose of MTX causing50% growth inhibition in comparison to control cells increasedfrom 3x10^–2 µM for MTX^s AT5BI-VA cells to 250 µMMTX for the MTX^r AX100 subclone. In contrast, human skin fibroblastsof healthy individuals did not show DHFR gene amplificationand loss of SV40 sequences under comparable conditions and wereunable to grow in MTX concentrations >1 µM. Gene amplificationand loss of DNA sequences are features underlying the genomicinstability known to be a characteristic property of A-T cellsand being probably responsible for the high cancer incidencein these patients.     

Loss of heterozygosity for the short arm of chromosome 11 (11p15) in human milk epithelial cells immortalized by microinjection of SV40 DNA.

We have developed, by microinjection of SV40 DNA into human milk epithelial cells, a new mammary cell line, Hu-MI, which exhibits the phenotype of luminal cells or so-called "breast cancer precursor cells." This cell line retains the phenotype of primary cells as demonstrated by the expression of keratins 18 and 19 and of polymorphic epithelial mucins. However, the cells do not grow in agar after more than 80 passages, nor do they form tumors in nude mice. Established cells contain 2 copies of SV40 DNA integrated into the cellular genome and up to 14 copies of free SV40 DNA. A deletion of the short arm of chromosome 11 (11p15) including the c-Ha-ras and the beta-globin genes was found in the immortalized cells when the DNA from these cells was compared to the DNA from peripheral blood mononuclear cells obtained from the same donor. In addition, this cell line showed a good transfection efficiency for other DNA sequences using classical transfection and selection techniques with a neomycin resistance gene (pKOneo). Selective microinjection of DNA into tumor precursor cells may prove useful for the study of the molecular mechanisms involved in breast carcinogenesis. The possible significance of the loss of 11p13-15 in malignant progression of breast cancer is discussed.     

Altered growth-regulation of prostatic epithelial-cells by human papillomavirus-induced transformation

Six lines of human papillomavirus (HPV)-transformed prostatic epithelial cells, clonally-derived from transfection of three different parental cell strains, were analyzed for their ability to respond to stimulatory and inhibitory factors known to regulate prostatic cell growth. The cell lines were tested in a serum-free medium that was developed specifically for analysis of low-density growth of prostatic cells in order to mitigate any effects of autocrine factors. This medium has been used previously to characterize the growth regulatory pathways of primary cultures of prostatic epithelial cells derived from normal, benign prostatic hyperplasia (BPH), or adenocarcinoma tissues, as well as an SV40-transformed cell line (pRNS-1-1) that was derived from the same parental cell strain as four of the HPV-transformed lines used in the present study. The responses of the HPV-transformed cell lines to three essential mitogens in the medium - epidermal growth factor (EGF), bovine pituitary extract (BPE) and insulin-like growth factor (IGF) - were similar to those of primary cultures and pRNS-1-1, except that two of the HPV-transformed cell lines were IGF-independent for growth. The presence or absence of cholera toxin (CT), which acts synergistically with peptide growth factors, only mildly affected the proliferation of HPV-transformed cells, which is also true for primary cultures and pRNS-1-1. However, while hydrocortisone (HC) also has only minimal effect on the growth of primary cultures and pRNS-1-1, four of the HPV-transformed lines were very dependent on HC for growth. With regard to growth-inhibitory factors, all of the cell lines (HPV- or SV40- transformed) were insensitive to tumor necrosis factor-alpha (TNFalpha), which is a common feature of transformed cells. However, the cell lines remained sensitive to the growth-inhibitory properties of retinoic acid (RA) and transforming growth factor-beta (TGF(beta)). The HPV-transformed cell lines were also inhibited by 1,25(OH)(2) vitamin D-3 [1,25(OH)(2)D-3], although pRNS-1-1 cells were not. Perhaps the most unexpected finding in our study was the apparent loss of responsiveness of all of the transformed lines to fibroblast growth factor (FGF). Alterations in FGF pathways have been described for prostatic cancer cell lines and changes in expression of FGF or receptors may be involved in prostatic carcinogenesis. Our study demonstrates that the availability of primary cultures, SV40- and HPV- transformed cell lines and a well-defined culture system will provide an opportunity to characterize the processes involved in malignant transformation of prostatic cells.     

Early superoxide dismutase alterations during SV40-transformation of human fibroblasts.

The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.     

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus.     

DNA damage stimulates human cell transformation by integrative but not episomal Epstein-Barr virus-derived plasmid

Previous work has demonstrated that ultraviolet (UV) irradiation of SV40-based plasmids can strikingly enhance the frequency of stable transformation of human cells. In this study we compared the effect of UV-induced DNA damage on transformation mediated by integrative versus autonomously replicating plasmids derived from human Epstein-Barr virus (EBV). We report that transfection of human fibroblasts with UV-irradiated integrative EBV-based plasmid results in enhanced transformation. However, transfection of UV-damaged episomal EBV-based constructs into the same human cell line does not enhance transformation; in fact, the extrachromosomal status of the plasmid is maintained irrespective of the UV dose to the plasmid. We conclude that enhanced transformation of human cells by damaged DNA requires its chromosomal integration.     

Decreased methylation rates of DNA in SV40-transformed human fibroblasts

The rates of methylation of total cellular DNA and newly synthesized DNA were measured in four unrelated SV40-transformed human fibroblast lines and in four control parent fibroblast lines. Rates of methylation of total cellular DNA were decreased by a factor of 1.8-2.3 in the transformed cells relative to control cells. Methylation was largely (75%-87%) restricted to newly synthesized DNA in control and transformed fibroblasts, and methylation rates of newly synthesized DNA were diminished in transformed cells by 12- to 19-fold relative to control cells. Growth rates were similar in the normal and transformed cells. The cellular uptake of methionine and conversion to S-adenosylmethionine were similar in the normal and transformed cells, suggesting no major differences between the normal and transformed cells in the cellular transport of methionine, methionine S-adenosyltransferase activity, or the intracellular concentrations of methionine and S-adenosylmethionine. The diminished rates of DNA methylation that we have observed suggest a possible mechanism for altered gene expression and growth control in transformed cells.     

Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts

The cytotoxic and cell kinetic effects of the epipodophyllotoxin 4,6-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyr anoside) (VP-16) in cultured mammalian cells are thought to relate to the induction of DNA damage, specifically DNA strand interruptions. In an effort to explore this relationship in human cells we have identified a VP-16-hypersensitive human cell system, namely an SV40-transformed fibroblast line (AT5BIVA) originally derived from an ataxia telangiectasia (AT) patient. Evidence is presented that enhanced VP-16 sensitivity may be a consistent in vitro feature at AT derived cells. However, the intrinsic sensitivity (DNA strand breaks per lethal hit quantitated by nucleoid sedimentation) was the same for AT5BIVA and a corresponding normal control, indicating that the AT cell line accumulated more drug-induced DNA damage during short-term VP-16 exposures. It is suggested that AT cells may have abnormal topoisomerase II activity. The cell cycle responses of normal and AT cells to VP-16 exposure were complex, with the generation of parasynchronous S phase populations and the accumulation of cells in G2. Differences in cell killing or DNA strand breakage between normal and AT cells could only be correlated with the magnitude and kinetics of the G2 retention phenomenon. In short, there are several similarities in the action of ionizing radiation and VP-16. We suggest that the sensitivity of cellular DNA to VP-16-induced DNA damage and the kinetics of the G2 delay may be useful parameters for predicting the survival probability of drug-treated human tumor populations.     

The response of ataxia-telangiectasia homozygous and heterozygous skin fibroblasts to neocarzinostatin

Skin fibroblast strains from patients with ataxia-telangiectasia(A-T) were recently reported to be hypersensitive to the antitumorantibiotic neocarzinostatin (NCS). In this study, the distinctintermediate degree of NCS sensitivity previously shown withtwo strains of A-T heterozygous fibroblasts was extended andconfirmed in an additional eight strains. A sensitivity baselinefor A-T heterozygous cells has thus been established and mayserve for the laboratory diagnosis of A-T heterozygotes, a cancer-pronepopulation. The response of A-T homozygous and heterozygouscells to NCS was further characterized by two molecular parameters,DNA repair synthesis and inhibition of DNA replication. Thepattern of dose response with regard to DNA repair synthesis,as assayed by the benzoylated naphthoylated DEAE cellulose chromatographymethod, was similar in normal, A-T homozygous and A-T heterozygouscells, although certain variability between strains was observedwith regard to the amount of repair incorporation. This findingcorrelates with a similar observation made with the same cellstrains following -irradiation. Inhibition of DNA synthesisfollowing NCS treatment was reduced in A-T homozygous cells,as compared to normal cells, but the "Inhibition resistant"component of DNA synthesis typically observed following treatmentwith low doses of X-rays or bleomycin was not observed withNCS. A-T heterozygous cells showed somewhat less inhibitionof DNA synthesis than normal cells following NCS treatment,although this difference was small and was not significant enoughto serve as an additional laboratory diagnostic aid. It is concludedthat the reduced inhibition of DNA synthesis, rather than reducedextent of DNA repair synthesis, correlates with the cellularhypersensitivity of A-T homozygous cells. This hypersensitivityseems to be observed primarily, if not exclusively, with DNAbreaking agents.     

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.