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1.
Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence. 相似文献
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Tumorigenicity and viral gene expression in rat cells transformed by Ad 12 virions or by the EcoRI c fragment of Ad 12 DNA 总被引:10,自引:0,他引:10
Several rat kidney cell lines transformed either by Ad 12 virions or by the EcoRI C fragment (left 16%) of Ad 12 DNA have been characterized with respect to viral DNA content, viral gene expression, and tumorigenicity. Virion transformed cells contained viral DNA sequences from most regions of the viral genome while EcoRI C fragment transformed cells contained only part of the C fragment. All cell lines transcribed DNA sequences from the left 6.7% (HindIII F fragment) of the genome while some lines transcribed sequences extending into the adjacent HindIII H fragment. Virus transformed and DNA transformed lines induced tumors in immunocompetent rats with equal efficiency. 相似文献
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Analysis of BK virus-transformed cells and BK virus-induced tumors by DNA-DNA reassociation kinetics
M. P. Grossi A. Corallini F. Poli A. Valieri G. Milanesi G. Barbanti-Brodano 《Archives of virology》1981,67(4):345-354
Summary BK virus (BKV)-transformed cells and BKV-induced tumors as well asin vitro derived tumor cell lines were all found to contain BKV DNA sequences when analysed by DNA-DNA reassociation kinetics. In transformed cells the number of viral genome equivalents per diploid cell genome (VG/CG) decreased with increasing generations. Likewise, single cell clones had a lower VG/CG than parental transformed cells. BKV-induced tumors had a high VG/CG. Tumor cells cultivatedin vitro and their clones had a lower VG/CG than BKV-induced tumors from which they were derived, suggesting a multiclonal origin of tumors. Hamster tumors induced by subcutaneous inoculation of BKV-transformed cells or tumor cell lines had a higher VG/CG than cells producing them. Variation in VG/CG is discussed in terms of cell selection depending on differentin vitro orin vivo conditions of cell growth. In some cases, however, the decrease in VG/CG most likely depends on loss of free viral sequences from transformed cells or tumors.With 2 Figures 相似文献
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The arrangement of integrated viral DNA is different in BK virus-transformed mouse and hamster cells 总被引:3,自引:0,他引:3
G Meneguzzi N Chenciner A Corallini M P Grossi G Barbanti-Brodano G Milanesi 《Virology》1981,111(1):139-153
Secondary mouse and hamster cells were transformed with BK virus (BKV) or with purified BKV DNA. Restriction analysis of viral sequences present in transformed cell lines yielded complex patterns, indicating the presence in most lines of more than one viral intergration/cell and the existence of a large number of available integration sites. In spite of this variability, however, the arrangement of integrated viral sequences had specific features in the two types of transformed cells. Tandem insertions of full-size BKV genomes, which were a constant feature of transformed mouse lines, were not found in hamster lines. These appeared to contain no complete, intact BKV genomes, but only genomes interrupted by the insertion event or tandem integrations of incomplete viral sequences. Traces of free, full-size viral DNA were observed in all transformed mouse lines, but not in hamster lines. One transformed hamster line, after 84 passages in culture, showed the presence of tandemly integrated, full-size BKV DNA, which was not found in cells at 14 passages after the appearance of the transformed phenotype. This was the only BKV transformed line from which virus could be rescued by fusion with permissive cells. These results suggest that BKV DNA integration occurs differently in hamster (nonpermissive) and mouse (semipermissive) cells, and support the idea that excision of integrated viral DNA occurs usually via homologous recombination. 相似文献
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Four cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22% of the HSV genome. Of five revertant clones selected for 3H-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to 1. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from 12 to 28%. The number of virus DNA fragments per cell ranged from 1 to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-1 transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone 139 is not entirely composed of the HSV-1 and HSV-2 common sequences. 相似文献
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Six different group C adenovirus transformed hamster cell lines were employed to produce tumors in hamsters. The sera from these animals were then used to immunoprecipitate [35S]methionine-labeled adenovirus induced tumor antigens from virus infected and transformed cells. Collectively, these sera detect 14 virus induced tumor antigens. Based upon the regions of the adenoviral genome present and transcribed in each of the transformed cell lines, an estimated position of the genomic map location responsible for the induction of each of the tumor antigens or viral early proteins was determined. These sera were also employed to follow the synthesis of the adenovirus proteins during productive infection and in transformed cells. Based upon this analysis the tumor antigens can be divided into two groups, early and delayed early proteins, depending upon the time after infection that a protein was synthesized and detected by immunoprecipitation. A comparison of the Ad2 and Ad5 early proteins produced in virus infected and transformed cells indicated that several proteins have different apparent molecular weights that are serotype specific but independent of the species of host cell employed. 相似文献
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S G Tsar'kov T A Posevaia T M Khizhniakova M M Garaev I F Barinski? 《Voprosy virusologii》1985,30(5):544-549
Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44). The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas. A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines. The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line). 相似文献
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Analysis of the growth properties and physical state of the human papillomavirus type 16 genome in cell lines derived from primary cervical tumors. 总被引:2,自引:0,他引:2
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We have established three cell lines from keratinizing and nonkeratinizing cervical carcinomas with distinct growth properties in vitro and in vivo. Each cell line contained human papillomavirus type 16 DNA sequences, but the lines differed in the physical state of the viral genome present in the cells. A high copy number of episomal human papillomavirus type 16 DNA sequences was detected in the TC-140 line derived from a keratinizing cervical cancer. This cell line had an aneuploid karyotype, did not grow in soft agarose, and formed benign cystlike nodules in nude mice, similar in morphology to well-differentiated areas of the primary tumor. Only integrated human papillomavirus type 16 sequences were detected in the TC-146A and TC-146B lines established from a nonkeratinizing large-cell cervical carcinoma. These cell lines exhibited reduced sensitivity to transforming growth factor-beta 1 and produced invasive, but not progressively growing, tumors in nude mice. These cell lines should complement existing in vitro models of cervical carcinogenesis and provide useful tools for understanding the importance of virus integration in the transformation process as well as the cellular and molecular basis for tumor progression. 相似文献
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Cytotoxic Effector Cells from Infectious Mononucleosis Patients in the Acute Phase Do Not Specifically Kill Epstein-Barr Virus Genome-Carrying Lymphoid Cell Lines 总被引:1,自引:0,他引:1
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We describe a study in which we investigated the cytotoxic activities of thymusderived (T) lymphocytes and natural killer cells against Epstein-Barr virus (EBV) genome-carrying lymphoid cell lines. Purified subpopulations of lymphocytes from eight patients with infectious mononucleosis and six healthy normal EBV-seropositive donors were tested. Enriched T-cells were obtained by passing purified whole blood lymphocyte preparations through human immunoglobulin-anti-immunoglobulin-coated glass bead columns. The cytolytic activity of effector cells was determined by the ability of these cells to lyse human target cells that were internally labeled with (51)Cr. These targets included cells from both EBV genome-carrying and EBV genome-negative lymphoid lines derived from malignant tumors, as well as from lymphocytes transformed in vitro by EBV, and were chosen to represent a wide spectrum of EBV-associated membrane antigens. We found that cytotoxic T-cells from patients with infectious mononucleosis showed no EBV-related specific cell killing per se, although a trend for increased killing of cell lines derived from spontaneous in vivo growing tumors, EBV genome carrying or not, was noted; however, this trend was not observed with cell lines derived from cord blood lymphocytes after EBV infection in vitro. In addition, our data suggest that natural killer cells may play an important role in controlling EBV infection in patients with infectious mononucleosis in the acute phase of the disease, particularly since T-cells (obtained after removal on immunoglobulin-anti-immunoglobulin columns of natural killer cells presumably bearing Fc receptors) were less efficient killers than whole blood lymphocytes; furthermore, lysis by whole blood lymphocytes was also greatest against cell lines derived from malignant tumors (as opposed to in vitro EBV-transformed cord blood lymphoid lines), irrespective of whether these targets were EBV genome positive or negative. 相似文献
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Observations that the major histocompatibility (B) complex is a determinant of the growth pattern of Rous sarcoma virus (RSV)-induced tumors raised the question as to whether control is exerted at the level of a v-src-determined, i.e., transformation-specific, function. To investigate this point, the tumor size scores and tumor profile indices of v-src-induced tumors were compared in two lines of chickens congenic for B complex genotypes. The finding that the growth patterns of tumors, induced by v-src DNA inoculation at 6 weeks posthatch, differ in these two lines establishes that the B complex exerts control over tumor growth at the level of a v-src-determined function. The potential importance of this control, in terms of the naturally occurring case of an avian sarcoma virus infection, is suggested by the observation that the patterns of tumor growth in a given congenic line are similar whether the tumors are induced by v-src DNA or by RSV. 相似文献
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In an attempt to gain insight into the mechanism of oncogenic transformation by BK virus (BKV), a human papovavirus, we have probed for BKV sequences in transformed hamster cells in which oncogenic transformation had occurred as a result of transfection by human tumor DNA positive for BKV sequences. Even though the sources of the transfecting DNA contained BKV sequences, the transformed hamster cells which arose from the transfection for the most part did not retain BKV sequences. In only one barely detectable case was BKV-specific DNA found associated with chromosomal DNA, and in only a small minority of the transformed cells was BKV DNA detected in the Hirt supernatant, indicating an episomal configuration. Even in these few cases where BKV sequences were present in an episomal form, altered migration on gels of some BKV-positive bands (compared to bands derived from cloned viral DNA) suggested deletions and rearrangements of BKV DNA. We employed several different probe methodologies for these studies, including nick-translation, random primer and a non-isotopic biotinylated probe which gave a sensitivity that could detect better than 0.01 copy of viral genome per diploid cell. We conclude that transformation by transfection with human tumor DNA does not require persistence of the BKV viral genome, suggesting that either BKV virus was irrelevant to original oncogenesis, in analogy with models proposed by others for herpesvirus oncogenesis. 相似文献
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R. Mäntyjärvi H. Sarkkinen S. Parkkinen A. Ryhänen H. Karjalainen K. Syrjänen S. Syrjänen 《Archives of virology》1988,100(1-2):17-25
Summary Cultures of primary fibroblasts of C57BL/6J mice were used as targets for transformation by bovine papillomavirus type 1 (BPV1) DNA. Although no foci were observed, several lines of transformed cells were established by subculturing. These immortalized cell lines had in vitro growth characteristics in high and low serum media and saturation densities typical of transformed cells. Karyotype analyses revealed extensive aneuploidic changes. In two of the three cell lines analyzed, viral DNA was present in monomeric episomal form, in the third cell line all viral sequences were found in the high molecular weight region of a Southern blot. Despite the transformed phenotype, only one of the cell lines was tumorigenic in nude mice at a low level. 相似文献
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State of viral DNA in BK virus-transformed rabbit cells 总被引:5,自引:0,他引:5
N Chenciner M P Grossi G Meneguzzi A Corallini R Manservigi G Barbanti-Brodano G Milanesi 《Virology》1980,103(1):138-148
Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued. 相似文献
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Hovhannisyan G Rapp A Arutyunyan R Greulich KO Gebhart E 《International journal of molecular medicine》2005,15(3):437-442
The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future. 相似文献
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Spontaneous and induced herpesvirus genome expression in Marek''s disease tumor cell lines. 总被引:6,自引:0,他引:6
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We incubated 31 newly established Marek's disease tumor cell lines at 41 degrees C for 48 h after subculturing and then examined them to determine the spontaneous rates of expression of viral internal antigen(s), viral membrane antigen(s), and virus isolation. All but two of the lines were isolated from tumors induced by clone-purified Marek's disease virus strain JM-10, GA-5, RB-1B, and BC-1A in nine different genetic strains of chickens with defined histocompatibility antigens. The line-to-line variations in the rates of spontaneous expression for the antigens or virus rescue were great, but the levels of expression were very low in most cases. The median rates of expression for viral internal antigen, viral membrane antigen, and virus isolation were 32, 8, and 2 positive cells per 10(5) cells, respectively (ranges, 0 to 20,280, 0 to 22,990, and 0 to 220 positive cells per 10(5) cells, respectively). The ratio of viral internal antigen expression to virus isolation was extremely variable and often high, whereas the ratio of viral internal antigen to viral membrane antigen expression was more consistent and generally low. The virus strain which induced the cell line influenced the level of virus genome expression, but the cell genotype did not. Cell lines transformed by JM-10 virus, which exhibited low oncogenicity, had significantly (p less than 0.01) higher rates of expression than cell lines transformed by CA-5 and RB-1B viruses, which exhibited high oncogenicity. Treatment with iododeoxyuridine or incubation at 37 degrees C induced increased rates of expression in most lines but not in all lines. The degree of enhanced expression was inversely proportional to the rate of spontaneous expression. 相似文献
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Summary Restriction mapping of polyoma virus DNA in mouse tumor cell lines gave patterns that varied with the cell line examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. (The cell lines were derived from tumors induced by polyoma virusin vivo and were propagated continuously in culture.) Two of the cell lines contained multiple copies of tandemly integrated virus genomes as well as free virus DNA molecules. Two other cell lines appeared to contain only integrated virus genomes arranged as tandem repeats. Based on restriction analysis with eleven different endonucleases, the virus DNA in one of the cell lines containing both free and integrated virus genomes was not detectably defective or hypermethylated. This is in contrast to most previously described polyoma virus transformed mouse cells. These virus genomes may, however, contain point mutations or unobserved rearrangements. The second cell line possessing free virus DNA molecules contained both nondefective and defective virus genomes. Most, if not all, defective virus genomes in this line were integrated. The two other cell lines possessing only detectable integrated virus DNA apparently contained only defective virus genomes. The defect in both cases was a small deletion (0.2 kb) encompassing 0.12 map units on the physical map of polyoma virus DNA, a region coding for the proximal part of the large T antigen. Moreover, in contrast to the cell lines with free and detectably nondefective virus DNA, the virus DNA was extensively methylated in cell lines containing only integrated and defective virus genomes.With 6 Figures 相似文献