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1.
J Virol ; 80(13): 6657-68, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16775353

ABSTRACT

Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.


Subject(s)
Cell Transformation, Viral , DNA Damage/drug effects , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Karyopherins/metabolism , Mutagens/toxicity , Receptors, Cytoplasmic and Nuclear/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/radiation effects , Animals , Cell Line, Transformed , Cell Transformation, Viral/drug effects , Cell Transformation, Viral/radiation effects , Cytoplasm/metabolism , Cytoplasm/virology , DNA Damage/radiation effects , Gamma Rays , Humans , Intranuclear Space/metabolism , Intranuclear Space/virology , Rats , Ultraviolet Rays , Exportin 1 Protein
2.
Int J Radiat Biol ; 80(10): 757-67, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15799621

ABSTRACT

PURPOSE: The mechanism by which ionizing radiation induces chromosomal rearrangements in mammalian cells has for long been a subject of debate. In order to dissect these events at a molecular level, we have studied the sequences involved in gamma irradiation-induced rearrangements. MATERIALS AND METHODS: An inverse polymerase chain reaction (PCR)-based methodology was used to amplify rearrangements that had occurred between one of four target regions (in or neighbouring the avian myelocytomatosis viral oncogene homologue (c-MYC), cyclin-dependent kinase inhibitor 1A (CDKN1A), fibroblast growth factor receptor 2 (FGFR2), or retinoblastoma 1 (RB1) genes) and sequences elsewhere in the genome, following gamma irradiation and subsequent incubation at 37 degrees C of normal human IMR-90 fibroblasts. RESULTS: The sequences of 90 such rearrangements, including both inter- and intra-chromosomal events, have been analysed. Sequence motifs (including DNA topoisomerase recognition sites) were not found to be consistently present either at or near rearrangement breakpoint sites. Statistical analysis suggested that there was significantly more homology between the sites of DNA rearrangement breakpoints than would be expected to occur by chance, however, most DNA rearrangements showed little or no homology between the interacting sequences. The rearrangements were shown to predominantly involve transcriptionally active sequences, a finding that may have significant implications for our understanding of radiation-induced carcinogenesis. CONCLUSION: The results obtained are difficult to reconcile with most models for ionizing radiation-induced chromosomal aberration formation, but are consistent with the Transcription-Based model.


Subject(s)
Chromosomes/radiation effects , DNA/radiation effects , Gamma Rays , Gene Rearrangement/radiation effects , Transcription, Genetic , Cell Transformation, Viral/genetics , Cell Transformation, Viral/radiation effects , Chromosome Breakage/genetics , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/radiation effects , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Genome , Humans , Molecular Sequence Data , Radiation, Ionizing , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/radiation effects , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Receptors, Fibroblast Growth Factor/radiation effects , Retinoblastoma/genetics , Retinoblastoma/metabolism , Sequence Homology, Nucleic Acid
3.
Radiats Biol Radioecol ; 42(5): 475-80, 2002.
Article in Russian | MEDLINE | ID: mdl-12449812

ABSTRACT

The cell tumorigenic ability and the cell clonogenicity in semi-solid medium of highly radioresistant variant cell line, PIC-20 (the progeny of djungarian hamster fibroblast cell line DX-TK- surviving acute exposure to 20 Gy of gamma-irradiation), were examined. In the absence of additional radiation, no differences between tested features of non-irradiated PIC-20 cells and parental DX-TK- cells were observed. On the contrary, after gamma-irradiation with high doses the essential differences in the properties of the examined cell lines were revealed. After exposure to 10 Gy the surviving fraction of PIC-20 cells was 20 times higher than that of the parental cells. Both irradiated and non-irradiated PIC-20 cells produced colonies of similar size. It is revealed that even after irradiation with doses of 5, 10 or 15 Gy, the PIC-20 cells kept their tumorigenicity as high as non-irradiated ones. In all these cases the 90-100% of animals had the tumour, with the average latent period of tumour appearance after inoculation being the same both for irradiated and non-irradiated PIC-20 cells. After irradiation of parental DX-TK- cells with the highest dose of 15 Gy, the amount animals with tumour decreased by 70% and the average latent period of tumour appearance increased fivefold as compared with that for non-irradiated DX-TK- cells. The data obtained indicate that PIC-20 is highly radioresistant cells, which are able to proliferate both in semi-solid medium and in an animal organism even after radiation exposure to high doses.


Subject(s)
Cell Survival/radiation effects , Cell Transformation, Viral/radiation effects , Fibroblasts/radiation effects , Radiation Tolerance , Animals , Cell Line , Clone Cells , Cricetinae , Culture Media , Gamma Rays , Radiation Dosage
4.
Radiat Prot Dosimetry ; 99(1-4): 241-3, 2002.
Article in English | MEDLINE | ID: mdl-12194295

ABSTRACT

Mutations induced after low dose ionising radiation exposure have been intensively analysed not only for radiation risk estimation but also for basic understanding of cellular responses. Human lymphoblastoid TK6-20C cells were irradiated with 100 mGy of X rays and mutation at the heterozygous thymidine kinase (TK) locus was selected by trifluorothymidine (TFT) resistance. Although the mutation frequency at the TK locus increased from 5.6 x 10(-6) to 7.4 x 10(-6), this increase was not statistically significant. However, molecular analysis of TK mutants exhibiting loss of heterozygocity (LOH) demonstrated a clear effect of such low dose IR exposure. Exposure to 100 mGy X ray increased the fraction of hemizygous-LOH from 10% to 42%. In previous experiments, a similar tendency in the increase of hemizygous-LOH was also observed in TK6 cells after exposure to a 2 Gy dose of X rays. This type of LOH can be considered as a result of end-joining repair of DNA double strand breaks.


Subject(s)
Loss of Heterozygosity/radiation effects , Mutagenesis , Thymidine Kinase/genetics , X-Rays , Cell Line , Cell Transformation, Viral/radiation effects , Humans , Papillomaviridae/genetics , Thymidine Kinase/deficiency
5.
Carcinogenesis ; 21(2): 205-11, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10657959

ABSTRACT

Radon exposure is associated with increased risk for bronchogenic carcinoma. Mutagenesis analyses have revealed that radon induces mostly multi-locus chromosome deletions. Based on these findings, it was hypothesized that deletion analysis of multiple radon-induced malignant transformants would reveal common mutations in chromosomal regions containing tumor suppressor genes responsible for malignant transformation. This hypothesis was supported by a previous study in which tumorigenic derivatives of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were established following irradiation with 30 cGy of high linear energy transfer radon-simulated alpha-particles. Herein, we describe the analyses of 10 additional tumorigenic derivative cell lines resulting from the irradiation of five additional independent BEP2D populations. The new transformants have common cytogenetic changes, including the loss of chromosome (ch)Y, one of three copies of ch8, one of two copies of ch11p15-pter and one of three copies of ch14. These changes are the same as those reported previously. Analysis of PCR-amplified short tandem repeats of informative loci confirmed the loss of heterozygosity (LOH) at 12 loci spanning the length of ch8 in cell lines from four of the total of eight irradiation treatments to date and the loss of chY in all cell lines (8 of 8). LOH analysis with a total of 17 informative loci confirmed loss on ch14 in transformants from seven of eight irradiation treatments and indicated a 0.5-1.7 cM region of common involvement centered around locus D14S306. No LOH was detected at any of the informative loci on ch11. The overall results support our stated hypothesis. Further studies are currently in progress to determine whether the ch8 and ch14 regions contain genes with tumor suppressor function in bronchial epithelial cells.


Subject(s)
Bronchi/chemistry , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 8/genetics , Cocarcinogenesis , Genes, Tumor Suppressor , Lung Neoplasms/genetics , Neoplasms, Radiation-Induced/genetics , Repetitive Sequences, Nucleic Acid , Alpha Particles , Aneuploidy , Animals , Bronchi/pathology , Bronchi/radiation effects , Bronchi/virology , Cell Line, Transformed/transplantation , Cell Transformation, Viral/radiation effects , Chromosome Aberrations , Chromosome Deletion , Chromosomes, Human/radiation effects , Chromosomes, Human, Pair 14/radiation effects , Chromosomes, Human, Pair 8/radiation effects , Epithelial Cells/chemistry , Epithelial Cells/pathology , Epithelial Cells/radiation effects , Epithelial Cells/transplantation , Epithelial Cells/virology , Genetic Predisposition to Disease , Humans , Loss of Heterozygosity , Lung Neoplasms/etiology , Mice , Mice, Nude , Neoplasm Transplantation , Papillomaviridae/physiology , Polymerase Chain Reaction , Radon , Y Chromosome/radiation effects
6.
Cancer ; 83(11): 2346-52, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9840534

ABSTRACT

BACKGROUND: Human papillomavirus (HPV) infection represents the most important risk factor for cervical carcinoma. Levels of expression of E6 and E7 transforming oncoproteins of high risk HPV genotypes (i.e., HPV-16 and HPV-18) have been linked specifically to the mitotic activity of cervical carcinoma and appear to be necessary for maintaining the malignant phenotype. However, E6/E7 viral proteins recently have been reported to be effective tumor rejection antigens in animal models and humans. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. However, although the physiologic and cellular changes associated with high doses of irradiation have been well documented it has been shown only recently that an increased synthesis of specific cellular proteins is observed after irradiation. In this study, the authors analyzed the effects of high doses of gamma irradiation on the expression of E6/E7 oncoproteins in HPV-16-infected cervical carcinoma cell lines. In addition, the effects of radiation on major histocompatibility complex (MHC) restriction elements also were studied. METHODS: The effect of high doses of gamma irradiation (i.e., 1250, 2500, 5000, and 10,000 centigray [cGy]) on the kinetics of E6/E7 oncoprotein expression in two HPV-16 positive cervical carcinoma cell lines (i.e., CaSki and SiHa) was evaluated by Northern blot analysis. In addition, the effect of radiation on the expression of MHC molecules also was studied by Northern blot and fluorescence activator cell sorter (FACS) analysis. RESULTS: Dose ranging from 1250 (sublethal) to 10,000 (lethal) cGy significantly increased the expression of E6/E7 oncoproteins as well as MHC Class I molecules in CaSki and SiHa cell lines when compared with untreated tumor cells. Both cell lines showed increased mRNA expression for MHC Class I molecules in a dose-dependent manner. E6/E7 oncoproteins also were up-regulated in a dose-dependent manner in the CaSki cell line, whereas in the SiHa cell line their expression plateau at 5000 cGy. When the kinetics of radiation-induced up-regulation of E6/E7 were studied, persistent up-regulation of the viral oncoproteins was noted for all doses of irradiation, with the lower and sublethal doses (i.e., 1250-2500 cGy) inducing the most significant enhancement. CONCLUSIONS: High doses of irradiation can induce a significant and long-lasting up-regulation of E6/E7 oncogenes and MHC Class I restriction elements on HPV positive cervical carcinoma cell lines. These effects by themselves suggest that irradiation could enhance local tumor immunogenicity in patients receiving radiation therapy. However, in contrast to this possible beneficial effect, sublethal tumor irradiation (up-regulating E6/E7 transforming oncoproteins) also could confer a significant growth advantage to radiation-resistant tumor cells. These findings, combined with the previously reported acquisition of a radiation-induced drug resistance, could provide a biologic basis for the poor prognosis of patients with cervical carcinoma recurrence after radiation therapy.


Subject(s)
Cell Transformation, Viral/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Genes, Viral , Oncogenes , Papillomaviridae/genetics , Uterine Cervical Neoplasms/genetics , Cell Transformation, Viral/radiation effects , Female , Gamma Rays , Histocompatibility Antigens Class I/radiation effects , Humans , Tumor Cells, Cultured , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology
7.
Tsitologiia ; 39(9): 809-21, 1997.
Article in Russian | MEDLINE | ID: mdl-9518387

ABSTRACT

Lymphoblastoid cell lines from patients with xeroderma pigmentosum (2 forms) and progeria (unusual form) were established using transformation of peripheral blood lymphocytes by Epstein--Barr virus. The influence of different UV doses on cell vitality, proliferation and cell cycle progression was studied by means of flow cytometry. The cell vitality was determined after incubation of cells with etidium bromide and FDA. We used cytograms with two logarithmic signals (log green/log red) to discriminate the cell cycle status. Cell cultures were used with density of 500,000 cells per 1 ml, previously synchronized at G-phase by the incubation in a medium with low serum content. The effect of UV irradiation was followed during 72 h. Among four analysed cell lines only line XP2SP demonstrated enhanced UV sensitivity, expressed by decreasing of the amount of living cells after the UV dose of 2.5 J/m2 and higher. The cell cycle studies showed that cells were blocked in S-phase and simultaneously the amount of apoptotic cells with both reduced DNA content and ability to bind FDA was seen increased. Similar events were observed in the control line only after the dose of 20 J/m2 and higher.


Subject(s)
Lymphocytes/cytology , Progeria/pathology , Xeroderma Pigmentosum/pathology , Cell Line , Cell Separation , Cell Survival/radiation effects , Cell Transformation, Viral/radiation effects , Cells, Cultured , DNA/radiation effects , DNA Repair/radiation effects , Dose-Response Relationship, Radiation , Flow Cytometry , Herpesvirus 4, Human , Humans , Lymphocytes/radiation effects , Microscopy, Fluorescence , Progeria/genetics , Ultraviolet Rays , Xeroderma Pigmentosum/genetics
8.
Leukemia ; 8(7): 1202-13, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8035613

ABSTRACT

The mechanism by which non-oncogene-bearing, slowly transforming retroviruses induce leukemia is not well understood, but appears to represent a multi-step process. Cell lines have been isolated following in vitro infection of lymphoid cells with radiation leukemia virus (RadLV) and they have been used to develop a two-step model for leukemia development. Thymic tumors were induced when one of the cell lines, C1-V13D, was inoculated into CBA/H mouse thymus. Upon reisolation of C1-V13D cells after one, two and three passages through thymus, individual cloned cell lines displayed increased tumorigenic potential compared with the non-tumorigenic parental line. Southern analysis has been used to track any genetic changes occurring while cells undergo further transformation and become increasingly tumorigenic. Specifically, retrovirus integration has been monitored in clones derived from C1-V13D at the primary, secondary and tertiary passage through thymus using probes specific for long terminal repeat (LTR), gag, pol and env genes of RadLV. The data indicate multiple ecotropic retrovirus integration sites in C1-V13D cells. Primary thymic tumors also showed the integration of a new recombinant or defective virus. There was no evidence that new ecotropic retrovirus integration had occurred during subsequent passage of primary tumors through the thymus, i.e. during the progression to oncogenesis. All data indicate an important role for the thymic environment in the development of a fully transformed cell.


Subject(s)
Cell Transformation, Viral/radiation effects , Leukemia, Experimental/genetics , Leukemia, Experimental/microbiology , Oncogenes/radiation effects , Radiation Leukemia Virus/genetics , Animals , Cell Division/physiology , DNA, Viral/genetics , Disease Models, Animal , Female , Male , Mice , Mice, Inbred CBA , Thymus Gland/cytology , Thymus Gland/microbiology , Thymus Neoplasms/microbiology , Thymus Neoplasms/pathology , Virus Integration
9.
Hum Genet ; 93(1): 21-6, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8270250

ABSTRACT

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with 60Co gamma-rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22-->qter or del(8)q23--> qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybrids, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1--> q11.1.


Subject(s)
Chromosomes, Human, Pair 8 , DNA Repair/genetics , Animals , Base Sequence , Blotting, Southern , Cell Fusion , Cell Line , Cell Transformation, Viral/radiation effects , Cells, Cultured , Chromosome Mapping , Cobalt Radioisotopes , DNA/radiation effects , DNA Repair/radiation effects , Fibroblasts , Humans , Mice , Mice, SCID , Molecular Sequence Data , Polymerase Chain Reaction , Radiation Tolerance/genetics , Simian virus 40 , Transfection
10.
Bioelectromagnetics ; 14(3): 215-28, 1993.
Article in English | MEDLINE | ID: mdl-8323572

ABSTRACT

Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 microT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced. This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36 degrees C transformed, 41 degrees C "revertant"). When grown at 36 degrees C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid "microfluidity." These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed.


Subject(s)
Adenosine Deaminase/metabolism , Cell Division/radiation effects , Cell Transformation, Neoplastic/radiation effects , Cell Transformation, Viral/radiation effects , Electromagnetic Fields/adverse effects , Environmental Exposure/adverse effects , Fibroblasts/radiation effects , Animals , Chick Embryo , Fibroblasts/enzymology
11.
Int J Radiat Biol ; 62(4): 461-8, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1357059

ABSTRACT

A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40 T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D0 = 192 +/- 13 vs 127 +/- 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. A retinoblastoma susceptibility gene (RB) binding defective mutant showed moderately increased radioresistance (D0 = 174 +/- 10). However, cell clones transfected with three different p53 binding defective mutants showed no change in radiosensitivity (D0 = 132 +/- 5) as compared with neo gene transfected controls. Transfection with T-antigen mutants defective in either the RB or p53 binding domain yielded no morphological alterations characteristic of transformation. These data suggest that the SV40 T/p53 complex may be of importance in the radioresistance phenotype.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , Cell Transformation, Viral/genetics , Genes, Retinoblastoma , Genes, p53 , Radiation Tolerance , Transfection , Antigens, Polyomavirus Transforming/chemistry , Cell Adhesion , Cell Line , Cell Survival , Cell Transformation, Viral/radiation effects , Colony-Forming Units Assay , Diploidy , Fibroblasts , Humans , Infant, Newborn , Male , Mutation , Skin/cytology
12.
J Leukoc Biol ; 50(4): 341-8, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1919362

ABSTRACT

Human T-cell cultures infected with human T-lymphotropic virus type I (HTLV-I) and interleukin-2 (IL-2)-dependent for their continuous growth were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then maintained in the medium containing phorbol 12-myristate 13-acetate (TPA). Cells achieved independence from IL-2 but became TPA-dependent for continuous growth. Multiple ultraviolet (UV) irradiations of TPA-dependent cells resulted in their autonomous growth. G-band karyotype analysis revealed multiple chromosomal abnormalities that were seen in cells before and after MNNG treatment and UV irradiations, and those that were only seen in autonomously growing cells. Viral expression was found to be transiently enhanced in association with emergence of certain chromosomal changes. Exposure of HTLV-I infected cells to certain mutagens may promote the occurrence of the specific rearrangement of cellular genes responsible for regulation of cellular and viral replication and may lead these cells to neoplastic transformation.


Subject(s)
HTLV-I Infections/drug therapy , Human T-lymphotropic virus 1/growth & development , Lymphocytes/microbiology , Methylnitronitrosoguanidine/pharmacology , Ultraviolet Rays , Cell Division/drug effects , Cell Division/radiation effects , Cell Transformation, Viral/drug effects , Cell Transformation, Viral/genetics , Cell Transformation, Viral/radiation effects , Cells, Cultured , Chromosomes/drug effects , Chromosomes/radiation effects , Chromosomes/ultrastructure , Combined Modality Therapy , DNA, Viral/drug effects , DNA, Viral/genetics , DNA, Viral/radiation effects , Female , HTLV-I Infections/radiotherapy , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/physiology , Humans , Karyotyping , Lymphocytes/drug effects , Lymphocytes/radiation effects , Male , Methylnitronitrosoguanidine/therapeutic use , Tetradecanoylphorbol Acetate/pharmacology
13.
Tsitologiia ; 33(5): 39-47, 1991.
Article in Russian | MEDLINE | ID: mdl-1796519

ABSTRACT

The kinetics of repair of the ionizing radiation-induced DNA single- and double-strand breaks in the normal NIH 3T3 mouse cells and in those transformed with virus oncogenes v-myc has been investigated. The incubation of non-transformed cells for 18 hours in serum-free medium results in significant decrease in the rate of the single-strand DNA breaks repair during the first minutes of post-irradiation incubation. This effect is absent in transformed cells. The DNA double-strand breaks repair is more efficient in transformed NIH 3T3 cells as compared to that in the non-transformed ones both after their incubation in the medium with 10% fetal bovine serum or without serum. However, more significant differences in the rate of elimination of these DNA lesions was found in the serum-free medium. Hence, the presence of v-myc sequences in the transformed cells prevented from a decrease in the efficiency of DNA repair due to incubation of cell culture in serum-free medium. These results agree with the assumption that c-myc gene product may be a mediator in regulation of DNA repair by the epidermal growth factor. These data also show that the c-myc gene expression in an important condition providing a high efficiency of the constitutive DNA repair process.


Subject(s)
3T3 Cells/radiation effects , Cell Transformation, Viral/radiation effects , DNA Damage/radiation effects , DNA Repair/radiation effects , DNA, Single-Stranded/radiation effects , DNA/radiation effects , Genes, myc/radiation effects , 3T3 Cells/metabolism , Animals , Cell Line, Transformed , Clone Cells/metabolism , Clone Cells/radiation effects , Culture Media , DNA/metabolism , DNA, Single-Stranded/metabolism , Mice , Time Factors
14.
Mol Carcinog ; 3(3): 141-9, 1990.
Article in English | MEDLINE | ID: mdl-2142598

ABSTRACT

We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.


Subject(s)
Adenoviridae/genetics , Cell Transformation, Viral/radiation effects , Adenovirus Early Proteins , Animals , Clone Cells , Cycloheximide/pharmacology , DNA Damage , DNA, Viral/analysis , Dactinomycin/pharmacology , Fibroblasts , Gamma Rays , Methyl Methanesulfonate/pharmacology , Oncogene Proteins, Viral/genetics , Rats
15.
Am J Otolaryngol ; 10(6): 399-403, 1989.
Article in English | MEDLINE | ID: mdl-2556945

ABSTRACT

We studied the production of heterophile antinuclear antibodies (ANAs) in the sera of 50 patients, 20 with nasopharyngeal carcinoma (NPC) and 30 with other head and neck cancers (laryngeal cancer and maxillary cancer), before and after radiation therapy. A higher incidence of ANAs was found in the sera of patients with NPC and ANA production in these patients was higher after radiation therapy. We therefore performed in vitro experiments to explore the mechanisms of ANA production in the serum of postirradiated NPC patients. X-ray-irradiated NPC-derived cells (NPC-KT) produced a large amount of Epstein-Barr virus (NPC EBV) compared with non-irradiated NPC-KT cells. Nasopharyngeal carcinoma EBV-infected lymphocytes produced high levels of ANAs. These data suggest that lymphocytes infected by EBV from NPC cells may produce ANAs in the sera of NPC patients.


Subject(s)
Antibodies, Antinuclear/analysis , Nasopharyngeal Neoplasms/pathology , Tumor Virus Infections/pathology , Cell Line , Cell Transformation, Viral/radiation effects , Fluorescent Antibody Technique , Herpesvirus 4, Human/pathogenicity , Humans , Nasopharyngeal Neoplasms/radiotherapy , Nasopharynx/pathology , Tumor Cells, Cultured/pathology , Tumor Virus Infections/radiotherapy , Virus Replication/radiation effects
16.
Mol Carcinog ; 2(5): 237-44, 1989.
Article in English | MEDLINE | ID: mdl-2557855

ABSTRACT

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.


Subject(s)
Cell Transformation, Viral , DNA Damage , Herpesvirus 4, Human/genetics , Cell Transformation, Viral/radiation effects , Cells, Cultured , Cloning, Molecular , DNA/radiation effects , DNA, Viral/genetics , Humans , In Vitro Techniques , Plasmids/radiation effects , Recombination, Genetic , Ultraviolet Rays
17.
Leuk Res ; 13(7): 629-32, 1989.
Article in English | MEDLINE | ID: mdl-2761294

ABSTRACT

Treatment of Friend erythroleukaemia cells with cytochalasin B (CB) resulted in multinucleation and loss of viability characteristic of a virus-transformed cell line. In an attempt to isolate pseudonormal revertants of this cell line mutagenized cultures were exposed to CB and surviving clones isolated. Many of these were found to be mutants resistant to the growth inhibitory effects of CB. The proportion of such mutants was reduced by simultaneous selection in CB and cytosine arabinoside. Of 699 clones examined none consistently exhibited reduced levels of multinucleation in the presence of CB. The inability of CB to select for revertants displaying a phenotype closer to normal cells is discussed.


Subject(s)
Cell Separation , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Viral/drug effects , Cytochalasin B , Leukemia, Erythroblastic, Acute/pathology , Tumor Cells, Cultured/pathology , Animals , Cell Line , Cell Separation/methods , Cell Transformation, Neoplastic/pathology , Cell Transformation, Neoplastic/radiation effects , Cell Transformation, Viral/radiation effects , Cytarabine/pharmacology , Cytochalasin B/pharmacology , Drug Resistance , Friend murine leukemia virus , Mice , Mutation , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effects , Ultraviolet Rays
18.
Mutagenesis ; 3(5): 389-95, 1988 Sep.
Article in English | MEDLINE | ID: mdl-3070272

ABSTRACT

Fibroblasts from patients with ataxia telangiectasia (AT) are hypersensitive to the lethal effects but hyposensitive to the mutagenic effects of ionizing radiation, suggesting that AT cells may be defective in some process which modifies damage to DNA. In this study we have examined the UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) of UV-irradiated adenovirus in AT fibroblasts. UVER was examined using both V antigen expression as well as progeny production from infected cell cultures. Viral mutagenesis was studied by examining the induction of phenotypically wild-type revertants among the progeny obtained from fibroblasts infected with a temperature-sensitive early mutant of adenovirus (Ad5ts36). UVER factors for Ad V antigen expression were significantly less than normal in the AT strains tested when infection occurred immediately after UV-irradiation of cells. However, UVER factors were greater than 1 and similar to those found for normal strains when cells were infected 24 h after UV-irradiation, indicating a delay in the expression of UVER for Ad V antigen in AT cells. UV-irradiation of both normal and AT cells 24 h prior to infection also resulted in a significant increase in progeny survival for UV-irradiated Ad. In normal cells, this progeny UVER was concomitant with a significant increase in the mutation frequency for UV-irradiated virus (increase in targeted mutagenesis) suggesting the existence of an inducible error-prone DNA repair mode in normal human cells. In contrast, pre-UV-irradiation of AT cells resulted in a significant decrease in the mutation frequency for UV-irradiated virus. These results suggest that AT cells lack an inducible error-prone DNA mode and that the delayed expression of UVER in AT cells results from a relatively error-free mechanism.


Subject(s)
Adenoviruses, Human/radiation effects , Mutation , Ultraviolet Rays , Adenoviruses, Human/genetics , Antigens, Viral/analysis , Ataxia Telangiectasia , Cell Survival/radiation effects , Cell Transformation, Viral/radiation effects , Cells, Cultured , Dose-Response Relationship, Radiation , Fibroblasts/cytology , Fibroblasts/radiation effects , HeLa Cells/cytology , HeLa Cells/radiation effects , Hemolytic Plaque Technique , Humans
19.
Mutagenesis ; 3(4): 297-8, 1988 Jul.
Article in English | MEDLINE | ID: mdl-2850445

ABSTRACT

The existence of untargeted viral mutagenesis in X-irradiated cells was investigated in a mammalian virus/cell system, where a low level of such viral mutagenesis can be demonstrated in UV-irradiated cells. In the positive control experiment UV-elicited mutagenesis was shown with cell exposures of 5, 10 and 15 J/m2 and a delay of 24 h between cell irradiation and infection with unirradiated herpes simplex virus. Although X-ray doses of 1, 3 and 10 Gy elicit enhanced reactivation of UV-irradiated virus, no untargeted mutagenesis for any X-ray dose at post-irradiation infection times of 0, 24 or 72 h was observed in this study. Thus untargeted mutagenesis of herpes simplex virus was not demonstrated in X-irradiated monkey cells, under conditions where X-ray-enhanced reactivation occurs and where untargeted mutagenesis in UV-irradiated cells occurs.


Subject(s)
Cell Transformation, Viral/radiation effects , Mutation , Simplexvirus/radiation effects , Animals , Cell Line , Chlorocebus aethiops , Dose-Response Relationship, Radiation , Kidney , Simplexvirus/genetics , Ultraviolet Rays , X-Rays
20.
Virus Res ; 9(1): 33-48, 1988 Jan.
Article in English | MEDLINE | ID: mdl-2829464

ABSTRACT

To investigate if Shope fibroma virus (SFV), a leporipoxvirus that induces benign tumors in adult rabbits, can trigger the second step of carcinogenesis in vitro or malignant transformation, an already immortalized rabbit cell line (SIRC) was inoculated with ultraviolet-irradiated virus. The resulting cell transformants displayed the characteristic properties of the malignant phenotype: lack of infectious particles, low serum requirement, high efficiency of cloning, resistance to superinfection, presence of viral DNA sequences in the nucleus, expression of viral proteins and induction of tumors in rabbits. However, this transformation was not stable since in all cell lines studied, a loss of the malignant phenotype was recorded close to the 50th passage. To assess the oncogenic potential of SFV, NIH 3T3 cells were transfected with SFV DNA. The results of these experiments indicate that SFV DNA can induce the formation of foci in certain NIH 3T3 cell lines. Taken together these results support the notion that SFV can elicit the transformation of cells in vitro.


Subject(s)
Cell Transformation, Neoplastic/etiology , Cell Transformation, Viral , Fibroma Virus, Rabbit/pathogenicity , Poxviridae/pathogenicity , Animals , Cell Line , Cell Transformation, Viral/drug effects , Cell Transformation, Viral/radiation effects , Cocarcinogenesis , DNA, Viral/genetics , Fibroblasts , Fibroma Virus, Rabbit/genetics , Fibroma Virus, Rabbit/radiation effects , Mice , Neoplasms, Experimental/etiology , Rabbits , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Ultraviolet Rays
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