Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.     

A Comparison of Survival and Repair of Uv-Induced DNA Damage in Cultured Insect VERSUS Mammalian Cells

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10–50 J/m² UV irradiations.     

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.     

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell‐killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual‐color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual‐color 143B human osteosarcoma cells, HT‐1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA‐1 human pancreatic cancer cells in vitro. UV‐induced cancer cell death was wave‐length and dose dependent, as well as cell‐line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m². With UVB irradiation, cell death was observed with irradiation at 50 J/m². For UVC, as little as 25 J/m² UVC irradiation killed approximately 70% of the 143B dual‐color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV‐induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection. J. Cell. Biochem. 110: 1439–1446, 2010. © 2010 Wiley‐Liss, Inc.     

PCR analysis of insertion site specificity, transcription, and structural uniformity of the Lepidopteran transposable element IFP2 in the TN-368 cell genome

The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.     

Effects of UV light on DNA chain growth and replicon initiation in human cells

Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. For rodent cells the extent and duration of this blockage is both cell line- and fluence-dependent. Using DNA fiber autoradiography we report here similar findings for human cells. Wild-type human cells did not exhibit significant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. Such activation would enable cells to replicate areas of DNA which do not contain a 'normal' site of initiation, yet contain blocking lesions both upstream and downstream. We have previously shown (Griffiths and Ling, 1987) that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. We report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation.     

Replication and passage of alfalfa looper nuclear polyhedrosis virus plaque variants in cloned cell cultures and larval stages of four host species

As alfalfa looper nuclear polyhedrosis virus (NPV) was serially passed through TN-368 cell cultures, the percentage of the cell population that developed infection with the MP variant decreased, while cells infected with FP variant increased. The MP variant was more virulent to Trichoplusia ni than the FP variant. Cells from the TN-368 cell line and strains derived from single cells were infected with isolated MP plaques. The viral progeny remained homogenous after one or occasionally two passages in cultured TN-368 cells. However, further serial passes in TN-368 cells or one pass in cell strains resulted in loss of the homogeneity, and the FP variant was detected. Four species of Lepidopterous larvae were also infected with the MP variant. Both variants were detected in viral progeny from diseased larvae.     

Differential requirements for baculovirus late expression factor genes in two cell lines.

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.     

The 3H-thymidine incorporation into G0 human lymphocytes induced by low doses of UVC radiation

The 3H-thymidine incorporation in human lymphocytes of healthy donors induced by UVC radiation under doses 0.0008-60 J/m2 was investigated. It was shown that the incorporation of 3H-thymidine increases under doses in interval 0.1-20 and is constant under doses higher than 20 J/m2. Under doses in interval 0.006-0.03 J/m2 near a half of all samples had the level of incorporation increased in comparison with control samples. We connect the presence, absence or variability of this effect with individual peculiarities of cells and with different activity of cell subpopulations that are different on morphological and physiological characteristics. The hypothesis about the role of this factor in the influence of low doses of pathogenic agents (UVC and X-radiation, chemical compounds) on human lymphocytes is discussed.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Multiple, small exposures of far-ultraviolet or mid-ultraviolet light change the sensitivity to acute ultraviolet exposures measured by cell lethality and mutagenesis in V79 Chinese hamster cells

Chinese hamster V79 cells (subline MI2G) were exposed repeatedly to fractionated doses of germicidal 254 nm light (far-uv) at 6 J.m-2/fraction/day or sunlight-simulating 290-330 nm (mid-uv) at 150 J.m-2/fraction/day and sensitivities to cell killing action and mutation of far-uv and mid-uv were examined. As the number of exposure fractions increased, the cell cultures became resistant to cell killing induced by both far-uv and mid-uv. Increases in both Do and Dq were observed. Treatment with exposures of 6 J.m-2 far-uv is more efficient in yielding cell cultures that are resistant than exposures of 150 J.m-2 mid-uv. In contrast to the cells exposed to repeated far-uv, the cells exposed to repeated mid-uv were relatively more resistant to cell killing effects of mid-uv than far-uv, suggesting a possible role of photolesions other than pyrimidine dimers. When mutants resistant to 6-thioguanine were assayed during repeated exposure to far- or mid-uv light, the yield was initially linear with accumulating dose. At high total accumulated doses, the frequency decreased gradually (6 J.m-2 mid-uv) or reached a plateau (150 J.m-2 mid-uv). The sensitivity of N80 cells (exposed to 80 fractions of mid-uv) to mutation induction by uv light is higher than that of the original MI2G cells, whereas U81 cells (exposed to 81 fractions of far-uv) have a sensitivity similar to that of the original cells. Although an initial decrease in resistance to cell killing was observed, resistant cells retained their characteristics after 100 days in culture without further exposure. Cross-resistance to X rays was not shown. The data in this paper suggest that the capacity for repair of photolesions in DNA by repair processes was enhanced in cell cultures by repeated exposure to far-uv or mid-uv and that this altered the cells' ability to cope with lethal and mutagenic lesions. It remains to be seen if these changes in cell sensitivity were brought about by selective or inductive processes or a combination of both.     

Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication

We are examining the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences (5 or 10 J/m2) of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV 40 DNA synthesis (incorporation of thymidine into viral DNA) in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication.     

The cytotoxicity of bleomycin A2 on adult rat liver epithelial cell lines at different stages of spontaneous cell transformation

Cytotoxic effects of Bleomycin A2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of (3H) thymidine for DNA biosynthesis, the incorporation rate of L-(3H) leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15-20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.     

Ultraviolet light exposure triggers nuclear accumulation of p21(WAF1) and accelerated senescence in human normal and nucleotide excision repair-deficient fibroblast strains

Induction of the p21(WAF1) protein (hereafter called p21) following genotoxic stress is known to inhibit proliferating cell nuclear antigen (PCNA)-dependent DNA repair, downregulate apoptosis, and trigger a sustained growth-arrested phenotype called accelerated senescence. Studies with immortalized human and murine cell lines have revealed that exposure to ultraviolet light (UVC; 254 nm) results in the degradation of p21 to facilitate DNA repair and promote cell survival, or may lead to apoptotic cell death. The objective of the present study was to determine whether exposure of non-transformed human fibroblast strains to relatively low fluences of UVC (i.e., fluences typically used in the clonogenic survival assay) might induce sustained nuclear accumulation of p21, leading to accelerated senescence. We have evaluated the responses of normal human fibroblast (NHF) strains and nucleotide excision repair (NER)-deficient fibroblast strains representing xeroderma pigmentosum (XP) complementation groups A and G and Cockayne syndrome (CS) complementation groups A and B. We report that exposure of NHFs to < or =15 J/m(2) of UVC, and NER-deficient fibroblasts to < or =5 J/m(2) of UVC, results in sustained nuclear accumulation of p21 and growth arrest through accelerated senescence. With each fibroblast strain examined, exposure to UVC fluences that resulted in approximately 90% loss of clonogenic potential triggered significant (>60%) accelerated senescence, but only marginal (<5%) apoptosis. We conclude that nuclear accumulation of p21 accompanied by accelerated senescence may be an integral component of the response of human fibroblasts to UVC-induced DNA damage, irrespective of their DNA repair capabilities.     

Establishment and characterization of a cell line from embryos of the Indianmeal moth,Plodia interpunctella

A cell line, UMN-PIE-1181, initiated in November, 1981, from embryos of a malathion-resistant strain of Indianmeal moth, Plodia interpunctella, was in the 83rd passage on January 28, 1985. The line consists of single, small, fibroblastlike cells that are polyploid with chromosome numbers ranging from 56 to 180. Growth rate is dependent on seeding density, there being no growth at or below seeding densities of $\text{2 ×}\text{10}{}^5\text{5}\text{,}\text{ml}$; optimum growth requires a fetal bovine serum concentration of at least 5%. Twenty-nine isozymes were examined. Five enzymes from the cell lines resolved well and subsequently were compared to enzymes extracted from 4-day-old embryos and other life stages of the insects. Phosphomannose isomerase, malic enzyme, malate dehydrogenase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase in extracts from the cultured cells and from the insects had identical patterns. Two bands for glutamate-oxalacetate transaminase, present in the cell line, were not observed in the tissue extracts. Furthermore, lactate dehydrogenase from the cultured cells appeared as four bands but was not detectable in any of the samples run from the various life stages of the insects.     

DNA double-strand break repair in eukaryotic cell lines having radically different radiosensitivities

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137Cs gamma rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.     

Recovery from exposure to DNA-damaging chemicals in radiation-resistant insect cells

Radioresistant TN-368 lepidopteran insect cells were examined with respect to their sensitivity to the chemical agents methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), propane sultone (PS), mitomycin C (MMC), and 4-nitroquinoline 1-oxide (4NQO). Based on survival ability, the TN-368 cells were more resistant than most mammalian cells to each of these agents. Concentrations of these agents which reduce survival to about 10% were used to assess recovery ability assayed by colony formation in liquid-holding and split-dose experiments. Liquid-holding experiments were performed by exposing cells in the plateau phase of growth for 1 h to 8 mM MMS, 50 microM MNNG, 9 mM PS, 110 microM MMC, or 175 microM 4NQO, removing the drug and incubating cells in spent medium for 6 h, and plating for colony formation. Split-dose experiments were performed by exposing exponentially growing cells to the above drug concentrations for 1 h, incubating in fresh medium for 6 h, exposing the cells to the agent for an additional hour, and plating. The TN-368 cells were able to significantly recover from MMS, MNNG and PS in both types of experiment. Recovery from 4NQO was observed in liquid-holding experiments and not assessed in split-dose experiments. In all cases where recovery was observed, survival enhancement was approximately 2-fold. Recovery from MMC (a cross-linking agent) exposure was not observed in either type of experiment. In addition, recovery from 8-methoxypsoralen plus UVA light (PUVA), another cross-linking treatment, was not observed. These studies indicate that DNA-DNA and/or DNA-protein crosslinking may be important molecular lesions causing death in the lepidopteran cells and that these cells may have some difficulty in repairing such damage.