Radiation enhanced reactivation of nuclear replicating mammalian viruses.

Abstract— When CV-1 monkey kidney cells were UV-irradiated (0–18 J/m²) or X-irradiated (0–10krads) before infection with UV-irradiated simian adenovirus 7 (SA7) or simian virus 40 (SV40), increases in the infectivity of these nuclear replicating viruses as measured by plaque formation were observed. These radiation enhanced reactivations, UV enhanced reactivation (UVER) and X-ray enhanced reactivation (X-ray ER), occurred both when virus infection immediately followed irradiation of the cells (except for X-ray ER with SA7) and when virus infection was delayed until 3–5 days after cell irradiation. While there was little difference in the levels of reactivation of UV-irradiated SV40 between immediate and delayed infection, delayed infection resulted in higher levels of reactivation of SA7. X-ray enhanced reactivation of UV-irradiated Herpes simplex virus persisted for several days but did not increase. Thus, X-ray enhanced and UV enhanced reactivations of these mammalian viruses were relatively long-lived effects. Essentially no UVER or X-ray ER was found in CV-1 cells for either immediate or delayed infection with UV-irradiated vaccinia virus or poliovirus, both of which replicate in the cell cytoplasm. These results suggest UVER and X-ray ER in mammalian cells may be restricted to viruses which are replicated in the cell nucleus.     

UV-ENHANCED VIRUS REACTIVATION IN MAMMALIAN CELLS: EFFECTS OF METABOLIC INHIBITORS*

Abstract—The induction process of UV-enhanced reactivation of UV-irradiated herpes simplex virus was investigated in CV-1 monkey kidney cells. A protein synthesis inhibitor, cycloheximide (0.5–5 μg/m/), present in the culture medium For 24 h between cell irradiation and virus infection decreased the enhanced virus survival normally found in UV-irradiated cultures. The enhanced virus reactivation became essentially resistant to the addition of cycloheximide by 6–8 h after cell irradiation, indicating that the cycloheximide-sensitive process necessary for enhanced reactivation was complete by that time. Since cycloheximide not only inhibits protein synthesis, but DNA synthesis as well, we investigated the effect of a DNA synthesis inhibitor, hydroxyurea. Hydroxyurea did not decrease UV-enhanced virus survival, but resulted in enhanced virus survival even in unirradiated cells. Therefore, the cycloheximide-caused inhibition of UV-enhanced reactivation did not arise from inhibition of DNA synthesis. The combined results indicate that (1) UV-enhanced virus reactivation in monkey kidney cells requires de novo protein synthesis during the first 6–8 h after cell irradiation and that (2) DNA synthesis inhibition may be the initiating event.     

UV-ENHANCED REACTIVATION OF MINUTE-VIRUS-OF-MICE: STIMULATION OF A LATE STEP IN THE VIRAL LIFE CYCLE

Abstract— UV-enhanced reactivation of minute-virus-of-mice (MVM), an autonomous parvovirus, was studied in parasynchronous mouse A9 cells. The survival of UV-irradiated MVM is increased in cells which have been UV-irradiated prior to infection. UV-enhanced reactivation can be explained neither by facilitated plaque detection on UV-treated indicator cells, nor by altered kinetics of virus production by UV-irradiated cells. No effect of the multiplicity of infection on virus survival was detected in unirra-diated or irradiated cells. The magnitude of UV-enhanced reactivation is a direct exponential function of the UV dose administered to the virus while virus survival is inversely proportional to the UV dosage. The expression of UV-enhanced reactivation can be activated in cells arrested in G₀, it requires de novo protein synthesis and it is maximal when cells are irradiated 30 h before the onset of viral DNA replication. Early phases of the viral cycle, such as adsorption to cellular receptors, migration to the nucleus and uncoating, were not affected by cell irradiation and are unlikely targets of the UV-enhanced reactivation function(s). These results, together with the single-strandedness of the viral genome, strongly suggest that the step stimulated in UV-irradiated cells functions concomitant with, or subsequent to, viral DNA replication.     

UV-ENHANCED REACTIVATION IN MAMMALIAN CELLS: INCREASE BY CAFFEINE

Abstract— It has been reported that caffeine decreases UV-enhanced reactivation of UV-irradiated Herpes simplex virus in CV-1 monkey kidney cells. That occurred when there was no delay between cell irradiation and virus infection. In the present study, virus infection was delayed following cell irradiation to allow an 'induction'period separate from the 'expression'period which occurs during the virus infection. Thus, the effects of caffeine on 'induction'and 'expression'could be determined separately. Caffeine increased the expression of UV-enhanced reactivation, while causing a small decrease in the 'induction'of enhanced reactivation.     

REACTIVATION OF UV- AND γ-IRRADIATED HERPES VIRUS IN UV- AND X-IRRADIATEDCV–1 CELLS

Abstract— Enhanced reactivation of UV- and y-irradiated herpes virus was investigated by the plaque assay onCV–1 monkey kidney monolayer cells irradiated with UV light or X-rays. Both UV- and X-irradiatedCV–1 cells showed enhancement of survival of UV-irradiated virus, while little or no enhancement was detected for y-irradiated virus assayed on UV- or X-irradiated cells. The enhanced reactivation of UV-irradiated virus was greater when virus infection was delayed 24 or 48 h, than for infection immediately following the irradiation of cells. It is demonstrable that the UV- or X-irradiatedCV–1 cells are able to enhance the repair of UV damaged herpes virus DNA, but not of y-ray damaged ones.     

ULTRAVIOLET-ENHANCED REACTIVATION OF HERPES VIRUS IN HUMAN TUMOR CELLS

Abstract —Virus-host cell interactions may be investigated by study of the enhancement of infectivity of ultraviolet (UV)-irradiated virus obtained by UV-irradiating the host cell [ultraviolet reactivation (UVR)]. This phenomenon was studied with Herpes simplex virus and normal (embryonic lung) and malignant (HeLa) human cells. Although the lung cells displayed no UVR, both the HeLa cells and a Sendai-virus carrier culture of HeLa cells demonstrated UVR capabilities. This UVR persisted at equal or increased levels for at least 24 h. Since the lung cells and HeLa cells have similar host-cell-reactivation (HCR) abilities, the differences in UVR suggests that UVR and HCR may operate by different mechanisms.     

HOST CELL REACTIVATION IN MAMMALIAN CELLS-V. PHOTOREACTIVATION STUDIES WITH HERPES VIRUS IN MARSUPIAL AND HUMAN CELLS

Abstract— The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovan cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes virus in Potoroo cells indicated a high level of "dark" host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (Λ > 600 nm) and human cells with normal repair and with ceils deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreacti-vating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps.     

HOST CELL REACTIVATION BY FIBROBLASTS FROM PATIENTS WITH PIGMENTARY DEGENERATION OF THE RETINA

–Cockayne syndrome (CS) is an autosomal recessive disease characterized by numerous clinical abnormalities including acute sun sensitivity and primary pigmentary degeneration of the retina. Cultured fibroblasts from CS patients are hypersensitive to ultraviolet (UV) radiation. Since host cell reactivation of irradiated virus is a useful probe to evaluate repair in different host cells, we studied such host cell reactivation in CS and in other diseases with retinal degeneration. The survival of UV-irradiated Herpes simplex virus type 1 was determined in fibroblast lines from four normal donors. two patients with CS, one with both xeroderma pigmentosum (XP) and CS, and from several other patients with (Usher syndrome, olivopontocerebellar atrophy, retinitis pigmentosa) and without (XP, ataxia telangiectasia) primary pigmentary degeneration of the retina. The viral survival curves (log survival vs linear fluence) in all cell lines showed two components: a very sensitive initial component (not quantitated in this study) followed by an exponential, less sensitive component. The exponential component had greater sensitivity than normal in the case of the CS patients, the patient with both XP and CS. and the XP patient. We propose that patients with CS have defective repair of DNA which may be the cause of their retinal degeneration.     

HOST-CELL REACTIVATION OF NON-LETHAL ULTRAVIOLET-EFFECTS

Abstract— Delay of intracellular growth of u.v.-irradiated bacteriophage T1 and Λ was compared in host-cell reactivating [HCR(+)] and non-host-cell reactivating [HCR(—)] bacterial strains. At a given phage survival level, intracellular growth delay occurs to the same extent in HCR (+) and HCR (-) strains; at a given absolute u.v.-dose, this delay is considerably more expressed in HCR (-) than in HCR (+) strains. Therefore, it does not reflect the time required for the HCR repair of otherwise lethal U.V. lesions. The results rather suggest that U.V. causes, besides lethal lesions, stable photoproducts in the DNA, which are a priori non-lethal, and which are recognized and efficiently eliminated by the HCR repair system. The HCR enzymes likewise act on (non-lethal) u.v.-photoproducts causing prophage induction in lysogenic cells. Consequently, one obtains the maximum induction effect in a lysogenic HCR (-) strain at a much lower u.v.-dose than in the corresponding lysogenic HCR (+) strain. In contrast, u.v.-damage causing loss of the host cell's capacity to support growth of unirradiated phage is not affected by HCR.     

CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/10T1/2 MOUSE FIBROBLASTS

C3H/10T1/2 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/m2 or mock irradiated and 24 h later infected with UV-irradiated Herpes simplex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.     

THE EFFECTS OF ULTRAVIOLET LIGHT ON HOST CELL REACTIVATION AND PLAQUE SIZE OF Herpes simplex VIRUS TYPE I IN C3H/lOT1/2 MOUSE CELLS

— Herpes simplex virus — type 1 (HSV-I) plaque-forming ability and plaque size were measured on C3H/1OT1/2 cell monolayers as functions of pretreatment dose with UV light at different times before inoculation with virus, in order to determine if UV-enhanced reactivation (ER) of UV-irradiated virus. as well as associated phenomena, could be obtained in this cell system. The number of virus plaques observed (i.e. the capacity of the cells to support virus growth) and the size of the plaques were found to increase substantially with pretreatment of the cells with UV light. However, no significant ER was observed. Therefore, the mechanisms responsible for the increases in plaque size and cell capacity seem to be independent of those responsible for ER. In work by others. C3H/l0T1/2 cells have hcen transformed by UV light at doses similar to those used in this study; the absence of ER of UV-irradiated virus in this study indicates that the mechanism underlying ER is not required for transformation.     

PHOTODYNAMIC TREATMENT OF HERPES SIMPLEX VIRUS INFECTION IN VITRO

Abstract— The effects of photodynamic action on in vitro herpes simplex virus infections of CV-1 monkey kidney fibroblasts or human skin fibroblasts were determined using proflavine sulfate and white fluorescent lamps. Photodynamic treatment of confluent cell monolayers prior to virus infection inactivated cell capacity, i.e. the capacity of the treated cells to support subsequent virus growth as measured by plaque formation. The capacity of human cells was more sensitive to inactivation than the capacity of monkey cells when 6 μM proflavine was used. Treated cell monolayers recovered the capacity to support virus plaque formation when virus infection was delayed four days after the treatment. Experiments in which the photodynamically treated monolayers were infected with UV-irradiated virus demonstrated that this treatment induced Weigle reactivation in both types of cells. This reactivation occurred for virus infection just after treatment or 4 days later. A Luria-Latarjet-type experiment was also performed in which cultures infected with unirradiated virus were photodynamically treated at different times after the start of infection. The results showed that for the first several hours of the virus infection the infected cultures were more sensitive to inactivation by photodynamic treatment than cell capacity. By the end of the eclipse period the infected cultures were less sensitive to inactivation than cell capacity. Results from extracellular inactivation of virus grown in monkey cells at 6 μM proflavine indicated that at physiological pH the virus has a sensitivity to photodynamic inactivation similar to that for inactivation of cell capacity. The combined data indicated that photodynamic treatment of the cell before or after virus infection could prevent virus growth. Thus, photodynamic inactivation of infected and uninfected cells may be as important as inactivation of virus particles when considering possible mechanisms in clinical photodynamic therapy for herpes simplex.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The effect of UV radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV-irradiated mammalian virus was tested. An action spectrum for radiation enhanced reactivation (RER) is presented. The shape of the action spectrum points to a combined nucleic acid-protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in RER does not allow us to distinguish which macromolecule is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.     

Expression of integrin β1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells

To investigate expression of integrin β₁ and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G₁ and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin β₁ on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin β₁ in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G₂+M phase, 11%; G₁ phase, 54%; S phase, 36%; the synchronous rates of G₁ and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin β₁ in G₁ phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G₁ phase HCC to HUVEC was significantly lower than the value of S phase cells (P<0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin β₁ was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G₁ and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin β₁ expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin β₁ played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.     

THE EFFECT OF METABOLIC INHIBITORS ON THE LARGE PLAQUE EFFECT WITH HERPES SIMPLEX VIRUS

Abstract— Herpes simplex virus (HSV) macroplaque strain plaque development is faster on ultraviolet (UV)-irradiated African green monkey kidney cells if viral infection is delayed for 12 h or more after cell irradiation (Coohill et al. , 1980). This phenomenon has been termed the large plaque effect (LPE). Here we show that treatment of UV-irradiated cells with cycloheximide inhibits the LPE. Pretreatment of unirradiated cells with hydroxyurea, caffeine, or acetoxy-acetylaminofluorene results in faster plaque development. Treatment of UV-irradiated cells with either hydroxyurea or caffeine gave a LPE of the same magnitude as UV alone. In addition, the LPE was observed with other HSV strains—microplaque, syn-20, and KOS. Our results are consistent with the interpretation that the LPE is 'inducible' in African green monkey kidney cells and that inhibition of DNA synthesis is the inducing event. Possible causes of the LPE and similarities between the LPE and enhanced viral reactivation are discussed.     

THE EFFECTS OF HOST-CELL REACTIVATION ON ASSAY OF U.V.-IRRADIATED HAEMOPHILUS INFLUENZAE TRANSFORMING DNA

Abstract— The host cell reactivation (HCR) mechanism in Haemophilus influenzae cells is inhibited by sub-microgram concentrations of acriflavine (as is already known to be true for Escherichia coli ). Exposure of these cells to similar concentrations of the drug during bacterial transformation increases the apparent ultraviolet light (u.v.) sensitivity of previously irradiated transforming DNA, indicating a repair of this DNA after uptake by the cells under normal conditions. Repair is inhibited by applying acriflavine at any time up to one hour after competent cells contact the irradiated transforming DNA. The fraction of the u.v. damage repaired by HCR is very different for different genetic markers. Those markers which are most u.v. sensitive when assayed in the absence of acriflavine are most poorly repaired, suggesting that this is the reason for their higher sensitivity. For all markers the fraction of the damage repairable by in vitro photoreactivation is approximately constant, and strongly overlaps the damage repairable by HCR. The degree of HCR achieved can be altered by experimental treatment of the H. influenzae DNA prior to transformation. Thus treatment of irradiated DNA with an enzyme from Micrococcus lysodeikticus –known to attack u.v. damaged, but not undamaged DNA–prevents subsequent intracellular repair of the same u.v. lesions whose repair is inhibited by acriflavine. Similarly, partial replacement of the thymine in transforming DNA by 5-bromouracil (BU) strongly inhibits repair of subsequent u.v. damage. As in bacteriophage, the BU effect is relieved if the u.v. exposure occurs in the presence of cysteamine. It is clear that intracellular repair must be considered in interpreting experiments with u.v.-irradiated transforming DNA.     

OVERLAPPING ACTION OF HOSTCELL REACTIVATION AND PHOTOREACTIVATION IN BACTERIA

Abstract— The photoreactivation rate of U.V. irradiated phages is decreased in u.v. irradiated bacteria. In contrast, the normal photoreactivation rate is observed if the irradiated bacteria are photoreactivated before phage infection. The decrease of the photoreactivation ratc is understood as a competing effect of the u.v. lesions in the bacterial nucleic acids for the photoreactivation enzyme. This competitive inhibition can be diminished not only by photoreactivation of the bacteria before phage infection but also by hostcell reactivation of the u.v. lesions in the bacterium. The results provide strong evidence that hostcell reactivation and photoreactivation revert the same u.v. photoproducts in bacterial nucleic acids. The experiments show that the hostcell reactivation enzyme is not induced by phage infection or by irradiation, but is normally present in the bacterial cell.     

Effects of isothermal 2.45 GHz microwave radiation on the mammalian cell cycle: comparison with effects of isothermal 27 MHz radiofrequency radiation exposure

Synchronized Chinese hamster ovary (CHO) cells were exposed to continuous wave (CH) 2.45 GHz microwave radiation (MWR) or CW 27 MHz radiofrequency radiation (RFR) under isothermal conditions (37±0.2°) to test the following hypotheses: (1) high frequency electromagnetic radiation exposure directly affects the mammalian cell cycle in the absence of radiation-induced heating; and (2) the magnitude of the cell cycle alteration is frequency dependent. CHO cells in either G₀/G₁-, S−, or G₂/M-phase of the cell cycle were simultaneously exposed to CW 27 MHz RFR or CW 2.45 GHz MWR at specific absorption rates (SARs) of 5 or 25 W kg⁻¹, or sham exposed, at 37±0.2°C. Cell cycle alterations were determined by flow cytofluorometry over a 4 d period after exposure. The DNA distributions of RFR, MWR, and sham exposed cells were compared to detect qualitative effects on the cell cycle. Quantitative measures of the effects of isothermal radiation exposure were determined from differences in the number of exposed and sham exposed cells in various cell cycle phases as well as comparison of the mean DNA content of exposed and sham exposed cell samples. Flow cytofluorometric assay precision and accuracy were determined by comparison of DNA distributions of replicate CHO control cell samples and by the use of internal DNA standards.Exposure to 27 MHz RFR or 2.45 GHz MWR altered the CHO cell cycle for periods of up to 4 d following exposure at SARs of 5 or 25 W kg⁻¹. There were significant differences in temporal responses, cell cycle phase sensitivity, and overall degree of cell cycle alteration for 27 MHz compared with 2.45 GHz radiation exposure. In contrast to the effect of 27 MHz RFR, which did not affect G₂/M-phase CHO cells, 2.45 GHz MWR altered all cell cycle phases to varying degrees. Exposure to 2.45 GHz MWR at 5 or 25 W kg⁻¹ was twice as effective as 27 MHz RFR in inducing cell cycle alterations as determined by differences in the number of exposed versus sham-exposed cells in various cell cycle phases.     

纳米Fe3O4-TiO2靶向光动力疗法对肝癌细胞的杀伤效应

光动力疗法(PDT)作为一种迅速发展的传统替代疗法,在抗癌治疗中显示出巨大的潜力.为增强靶向性和提高光催化杀伤效率,本研究设计了一种新型光敏剂Fe₃O₄-TiO₂磁性纳米粒.在不同外磁场下,考察其在可见光和紫外光激发下对肝癌细胞的杀伤效应.同时利用流式细胞术检测纳米Fe₃O₄-TiO₂对肝癌细胞凋亡率、细胞周期和线粒体膜电位的影响.根据纳米Fe₃O₄-TiO₂和肝癌细胞的作用方式探讨其抗癌机制.结果表明,可见光激发纳米Fe₃O₄-TiO₂可以杀伤癌细胞,且其杀伤效率与紫外光激发下无明显差别.此外,Fe₃O₄-TiO₂比TiO₂具有更高的细胞摄取率,从而使其具有更高的选择性和光催化杀伤效率.其作用机制是光催化纳米Fe₃O₄-TiO₂产生活性氧ROS抑制癌细胞,然后通过阻滞细胞周期G₀/G₁期,降低线粒体膜电位,线粒体去极化,最终诱导细胞凋亡.