UV Irradiation induces an activity which stimulates Simian virus 40 rescue upon cell fusion.

UV irradiation of African green monkey cells greatly stimulated efficiency of simian virus 40 induction from simian virus 40-transformed Syrian hamster cells after cell fusion. The maximum inducing activity was observed at 15 to 20 h after irradiation but remained only transiently. The addition of cycloheximide after UV irradiation eliminated the stimulation of the activity.

Effect of caffeine on induction of endogenous type C virus in mouse cells in vitro.

The effect of caffeine on the expression of murine endogenous virus in mouse cells induced by radiation and chemicals was studied. Postirradiation treatment of K-BALB cells with caffeine enhanced cell killing as well as the induction of xenotropic virus after ultraviolet light irradiation. The degree of enhancement for the virus induction was comparable to that for cell killing. On the other hand, colony-forming ability and the expression of xenotropic virus of K-BALB cells after X-irradiation were unaffected by caffeine. These data suggest a linear relationship between the degree of endogenous virus expression and the amount of lethal damages after irradiation. For induction by halogenated pyrimidines, a 24-hr incubation of AKR2B cells with caffeine after 5-iodo-2'-deoxyuridine treatment resulted in marked suppression of the expression of ecotropic virus. On the contrary, in K-BALB cells, caffeine exerted only a small effect on 5-iodo-2'-deoxyuridine-induced expression of ecotropic and xenotropic viruses. These results indicate that, although using the same inducing agent, the pathway of endogenous virus induction may be different for AKR2B cells and for K-BALB cells.

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions.

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.

Analysis of oncogenic progression in a radiation leukemia virus model.

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.

Autonomous growth of human T-lymphotropic virus type I infected human lymphocytes treated with N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet rays.

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.

The effect of 8-methoxypsoralen-plus ultraviolet light on cell-virus interaction: the transforming infection; effect of PUVA on the transformation of baby hamster kidney cells by polyoma virus.

Pre-treatment of baby hamster kidney (BHK) cells with 8-methoxypsoralen (8-MOP) plus ultraviolet (UV) light enhances the frequency of their transformation by polyoma (Py) virus. Of the doses tested, 0.5 microgram/ml 8-MOP plus 0.3 J/cm2 UV-light results in maximal (30-fold) stimulation of viral transformation. 8-MOP alone does not affect viral transformation and UV-light alone causes only a slight increase in the transformation frequency. Thus the drug and light act synergistically in promoting the effect. Treatment of BHK cells with drug plus light without Py infection does not lead to a transformed morphology. A drug-light combination (0.01 microgram/ml 8-MOP plus 1.2 J/cm2 UV) that inhibits cellular DNA synthesis to 75% of control at 28 hr after treatment results in a 6-fold stimulation of the transformation frequency.

Characterization of the transformation properties of Shope fibroma virus.

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.

An attempt to select pseudonormal revertants of Friend erythroleukaemia cells using cytochalasin B.

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.

SV40-induced transformation and T-antigen production is enhanced in normal and repair-deficient human fibroblasts after pretreatment of cells with UV light.

Human fibroblasts irradiated with UV light were infected with simian virus 40 and tested either for transformation or T-antigen production. At UV doses that allowed approximately 5-10% of the irradiated cells to survive, the number of surviving transformed colonies increased. This result was confirmed by testing for T-antigen 96 h post infection by means of indirect immunofluorescence. Since these results were obtained for a normal cell line as well as for two UV excision repair-deficient ones (XP groups A and D), it was concluded that excision repair functions cannot play a decisive role in the events leading to increased transformation and T-antigen production. It is proposed that the relative increase of transformation and T-antigen production is the expression of host functions which are induced by DNA damage threatening cell survival.

Failla Memorial lecture. The future of heavy-ion science in biology and medicine.

Interplanetary space contains fluxes of fast moving atomic nuclei. The distribution of these reflects the atomic composition of the universe, and such particles may pose limitations for space flight and for life in space. Over the past 50 years, since the invention of Ernest Lawrence's cyclotron, advances in accelerator technology have permitted the acceleration of charged nuclei to very high velocities. Currently, beams of any stable isotope species up to uranium are available at kinetic energies of several hundred MeV/nucleon at the Berkeley Bevalac. Recently, new areas of particle physics research relating to the mechanisms of spallation and fission have opened up for investigation, and it is now realistic to search for nuclear super-dense states that might be produced in heavy nuclear collisions. The heavy ions hold interest for a broad spectrum of research because of their effectiveness in producing a series of major lesions in DNA along single particle tracks and because of the Bragg depth ionization properties that allow the precise deposition of highly localized doses deep in the human body. Individual heavy ions can also interrupt the continuity of membraneous regions in cells. Heavy ions, when compared to low-LET radiation, have increased effectiveness for mammalian cell lethality, chromosome mutations, and cell transformation. The molecular mechanisms are not completely understood but appear to involve fragmentation and reintegration of DNA. Cells attempt to repair these lesions, and many of the deleterious effects are due to misrepair or misrejoining of DNA. Heavy ions do not require the presence of oxygen for producing their effects, and hypoxic cells in necrotic regions have nearly the same sensitivity as cells in well-oxygenated tissues. Heavy ions are effective in delaying or blocking the cell division process. Heavy ions are also strong enhancers of viral-induced cell transformation, a process that requires integration of foreign DNA. Some cell lines, known to be radioresistant to X rays, have exhibited greater sensitivity to heavy ions. These radiobiological properties, combined with the ability to deliver highly localized internal doses, make accelerated heavy ions potentially important radiotherapeutic tools. Other novel approaches include the utilization of radioactive heavy beams as instant tracers. Heavy-ion radiography and microscopy respond to delicate changes in tissue electron density. Dose localization with helium ions has achieved excellent results for pituitary tumors, tumors adjacent to the spinal cord, and ocular melanomas. We are working on adapting silicon- and neon-ion beams for controlled therapy studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains.

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.

Ionizing radiation-induced chromosomal rearrangements occur in transcriptionally active regions of the genome.

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.

Lack of enhanced mutation of UV- and gamma-irradiated herpes virus in UV-irradiated CV-1 monkey cells.

The frequency of forward mutation of unirradiated, UV-irradiated or gamma-irradiated herpes virus was determined after infecting UV-irradiated or unirradiated CV-1 monkey kidney cells, to investigate the correlation between UV-enhanced reactivation (UVER) and mutagenesis. UV-irradiation to cells had no effect on mutation frequency of irradiated virus even in the conditions in which UVER was maximally expressed for the survival of UV-irradiated virus.

Action spectrum for the in vitro induction of simian virus 40 by ultraviolet radiation.

A line of simian virus 40-transformed hamster kidney cells was exposed to ultraviolet radiation at eleven different wavelengths in the region 238-302 nm. An action spectrum derived from the resulting exposure-response curves for the induction of simian virus 40 from these cells exhibits a broad peak in the region 260-270 nm suggesting DNA as the major chromophore for this response. This conclusion is consistent with results obtained by other investigators who have noted viral induction by a number of DNA-damaging agents.

Further evidence that ultraviolet radiation-enhanced reactivation of simian virus 40 in monkey kidney cells is not accompanied by mutagenesis.

Can simian virus 40 (SV40) be used to detect mutagenic DNA repair in cultured mammalian cells? The published evidence from different laboratories are in direct conflict. In order to decide between the conflicting evidence, we conducted experiments in two separate laboratories using experimental protocols similar to those previously used to investigate mutagenic repair with viral probes. Mutagenesis in SV40 virus stocks obtained by infecting ultraviolet (UV)-irradiated or unirradiated CV-1 monkey kidney cells with UV-irradiated or unirradiated temperature-sensitive SV40 mutant tsB201 was investigated. The frequency of reversion of the ts mutant to phenotypically wild-type virus was determined by assaying the virus stocks at permissive (33 degrees) and non-permissive (39 degrees) temperatures. These data show that (a) the reversion frequency for unirradiated virus propagated in irradiated cells was more than that in unirradiated cells; (b) irradiated virus gave more reversion than unirradiated virus in unirradiated and irradiated cells; and (c) irradiated virus had a lower reversion frequency in irradiated cells than in unirradiated cells. Reactivation experiments carried out in parallel; with the mutagenesis showed enhanced reactivation in UV-irradiated SV40 in UV-irradiated CV-1 cells. We conclude that enhanced reactivation of UV-irradiated SV40 was not mutagenic in monkey kidney cells.

The effect of factors causing induction of DNA breaks on transfection of chicken cells by the XC DNA, and the kinetics of appearance of virus-producing cells after transfection.

The effect of BUdR and 4NQO treatment of the recipient chicken fibroblast cultures on the efficiency of transfection by the XC DNA was investigated. The efficiency of transfection was 2-fold higher when the recipient cultures were incubated in the presence of 10 micrograms BUdR/ml in medium 199 (48%) than when cultures were not treated (25%). The efficiency was not further increased by exposure of BUdR-treated cultures to visible light for 20 min (31%). Growth of BUdR-treated cultures decreased after light irradiation which indicated that BUdR was incorporated into the host cell DNA. Treatment of the recipient cultures with 4NGO at a concentration of 0.4 micrograms/ml for 2 h before transfection exerted only unfavourable effects on transfection efficiency. In cultures positive in transfection, transformation was first detected 3 days after exposure to DNA by the infectious centre assay, with a frequency of 4.76 +/- 6.46 transformed cells per 10(6) cells.

[Malignant cell transformation in rats in vitro as affected by the variolovaccine virus]. / Zlokachestvennaia transformatsiia kletok krys in vitro pod vliianiem virusa ospovaktsiny.

The capacity of the variolovaccine virus with low cytopathogenic activity to cause morphologic and malignant transformation of normal rat cells in vitro is detected. The transformed cells induced tumours in gamma-irradiated rats.

Mutation induction in human cells after low dose X ray exposure.

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.

[Retention of tumorigenicity in radioresistant progeny of cells surviving exposure to high doses of gamma irradiation]. / Sokhranenie tumorogennosti u radiorezistentnykh potomkov kletok, vyzhivshikh posle vozdeistviia gamma-izlucheniia v bol'shikh dozakh.

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.

[The isolation and analysis of lymphoblastoid cell lines from patients with xeroderma pigmentosum and progeria]. / Poluchenie i analiz limfoblastoidnykh kletochnykh linii ot bol'nykh pigmentnoi kserodermoi i progeriei.

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.