Environmental wavelengths of ultraviolet light induce cytoskeletal damage.

The ultraviolet component of sunlight is the major cause of skin cancer and is responsible for accelerating the aging of human skin. It is therefore important to determine the mechanisms by which ultraviolet light alters normal cellular functions. The potential importance of ultraviolet light-induced damage to non-DNA targets has received little attention. Since the cytoskeleton is an important participant in the control of normal cell growth, the microfilaments and microtubules of UV irradiated human skin fibroblasts have been studied using fluorescence microscopy. Polychromatic ultraviolet light, composed of environmentally relevant wavelengths, was found to disrupt the cytoplasmic microtubule complex in a dose dependent manner. The induction of microtubule disassembly did not correlate with the cytotoxicity of ultraviolet light of varying composition.

An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the coordinated reorganization of microtubules, microfilaments, and keratin intermediate filaments in cultured human epidermal keratinocytes following a switch from low-Ca++ (0.15 mM) medium to high-Ca++ (1.05 mM) medium. A dramatic reorganization occurs concurrently in the three major cytoskeletal components shortly after the calcium switch. The most prominent features are the alignment of keratin filaments at the plasma membranes of apposed cells, the induction of microfilament rings, the restriction of microtubules to the area within the boundaries of the microfilament rings, and the alignment of actin bundles at cell borders. Additional changes are observed in terminally differentiated cells. This is the first report that describes simultaneous changes in the organization of the three major cytoskeletal components of epidermal keratinocytes. Cytochalasin D and demecolcine (colcemid) studies were performed to determine whether the organization of microtubules, microfilaments, and keratin filaments, as well as the calcium-induced reorganization of these cytoskeletal elements, may be dependent on the existence of structural relationships between them. These studies demonstrate that the disruption of microfilaments results in the formation of a latticelike keratin network, with a close association of actin and keratin being maintained. The formation of keratin filament alignments occurs even in the absence of intact microfilaments. In addition, it was found that the Ca(++)-induced reorganization of microfilaments and keratin filaments is not dependent on an intact microtubule network. Furthermore, the reorganization of actin into concentric rings can be dissociated from changes in the organization of keratin filaments.

Repair of DNA damage induced in systemic lupus erythematosus skin fibroblasts by simulated sunlight.

Skin fibroblasts derived from three normal individuals and three patients exhibiting the disease systemic lupus erythematosus (SLE) were exposed to the simulated sunlight produced by a solar simulator. The induction and repair of DNA damage induced by this treatment were examined. The total number of lesions repaired by excision, as well as the removal of pyrimidine dimers and E. coli endonuclease III--sensitive sites did not differ significantly in the three SLE cell strains compared with normal cells. However, abnormalities in the formation and maintenance of DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) were found in SLE-4 and SLE-5 following simulated sunlight exposure. In contrast, SLE-3 cells exhibited responses similar to normal cells in reference to SSB and DPC formation. These findings correlate well with the previously determined UV sensitivity of these SLE cell strains.

Spontaneous transformation to anchorage-independent growth of a xeroderma pigmentosum fibroblast cell strain.

A skin fibroblast cell strain (GM2995) derived from a patient with xeroderma pigmentosum was received at low passage. As the cells were serially passaged (1:4 dilution), their size and growth characteristics changed. By passage 13, approximately 90% of the colonies produced by cells seeded at low density were composed of small, densely packed cells. Cells capable of anchorage-independent growth were observed after passage 7; they formed round, smooth-edged colonies in soft agar. The frequency of cells exhibiting anchorage-independent growth increased rapidly at subsequent passages, reaching 35-50% of the population by passage 20. This phenomenon was accompanied by the appearance of aneuploidy. These cells are still proliferating actively at passage 35. These late-passage GM2995 cells retain the extreme hypersensitivity to the cytotoxic effect of UV radiation characteristic of early-passage GM2995 cells.

Quantitation of herpes simplex virus in rabbit corneal epithelium.

The authors have developed an objective method for quantitation of herpes simplex virus in the corneal epithelium of rabbits. At appropriate times postinfection, full-thickness rabbit corneas were removed by trephination and subjected to one cycle of freezing and thawing. The corneal epithelium was then disrupted by sonication. The amount of infectious virus recovered from sonicated specimens was determined by an in vitro plaque assay, providing a measure of the quantity of virus present during the acute stage of herpetic keratitis. Using this technique, the authors found that the mean virus titer was reduced from 1.5 X 10(5) plaque forming units (pfu) per cornea in control rabbits to less than 200 pfu per cornea in rabbits treated topically for 2 days with 1% trifluridine. In contrast, instillation of 1% prednisolone acetate resulted in the persistence of higher levels of virus (275 pfu) than those observed in control rabbits (3 pfu) 4 days after the cessation of therapy.

Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes.

Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.

Varying sensitivity of human skin fibroblasts to polychromatic ultraviolet light.

The inactivation of normal and xeroderma pigmentosum cells has been compared following exposure to environmentally relevant wavelengths of ultraviolet light or to the more commonly investigated 254-nm ultraviolet light. The sensitivity of xeroderma pigmentosum cells to polychromatic long-wavelength ultraviolet light was greatly lower than that observed using 254-nm light. Such variations in the relative sensitivities of normal and xeroderma pigmentosum cells support the likelihood that the lesions which contribute to cellular alterations induced by solar wavelengths of ultraviolet light are different from those which predominate at 254 nm.

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains.

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis.

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.

Inhibition of postreplication repair and the enhancement of induction of SV40 virus from transformed hamster kidney cells.

The induction by ultraviolet light of simian virus 40 (SV40) from two SV40--transformed hamster kidney cell lines is enhanced by caffeine. In order to investigate the mechanism responsible for this enhancement, the effect of caffeine on postreplication repair of DNA damaged by UV light was studied utilizing alkaline sucrose-gradient sedimentation. Caffeine at concentrations of 0.5, 1.0 or 2.0 mM inhibited the filling of gaps during postreplication repair. In addition, caffeine was found to potentiate cell killing by mitomycin C, an alkylating agent, and to enhance SV40 induction by mitomycin C. We postulate that the persistence of gaps in DNA, caused by the presence of caffeine, results in the enhancement of SV40 virus induction.

Microtubule disassembly induced by sensitizing halogenated nitrobenzene derivatives.

The potency of certain halogenated nitrobenzene derivatives to induce allergic contact dermatitis has been demonstrated in guinea-pigs and in man. As the first step towards understanding the mechanisms of cellular injury induced by these sensitizers, we have studied by fluorescence microscopy the effects of 13 halogenated (nitro)benzene derivatives on microtubule organization in mouse 3T3 fibroblasts and human AG1522 skin fibroblasts. Untreated cells have numerous microtubules distributed in a network fashion throughout the cytoplasm and extending to the cell periphery. Exposing cells for 3 hr to micromolar doses of halogenated nitrobenzene derivatives, which induce allergic contact dermatitis in guinea-pigs and man, resulted in a dose-dependent disassembly of microtubules. In contrast, incubation with 10-70 times higher doses of seven halogenated (nitro)benzene derivatives that do not sensitize guinea-pigs or man, had no discernible effect on microtubule organization of both cell types under identical assay conditions. Thus, for the 13 halogenated (nitro)benzene derivatives tested in this study, a 100% positive correlation exists between the sensitizing capacity as determined by in vivo tests on guinea-pigs and man (Landsteiner and Jacobs, 1936) and the ability to cause microtubule disassembly in cultured cells. These results may provide the basis for developing an in vitro screening assay for identifying other potential halogenated nitrobenzene sensitizers. In addition, these studies represent a new approach to investigating the mechanisms of contact sensitivity induced by simple chemicals.

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts.

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.

Disruption of cytoplasmic microtubules by ultraviolet radiation.

Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts.

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.

The in vitro photosensitivity of systemic lupus erythematosus skin fibroblasts.

To investigate the role of DNA damage in the pathogenesis of systemic lupus erythematosus (SLE), we studied the ability of skin fibroblasts derived from SLE patients to recover from ultraviolet (UV) light radiation of varying wavelengths. Four of five SLE cell strains were more sensitive to UV-C (254 nm), sun lamp, and UV-A (320 to 400 nm) light than were normal cells. SLE cellular recovery was most sensitive to broad spectrum, long wavelength light. This hypersensitivity did not appear to result from the UV light activation of a clastogenic factor. Experiments which examined the DNA repair capacity of irradiated cells indicated that SLE fibroblasts may be able to excise certain DNA lesions as well as normal cells. The mechanisms responsible for the hypersensitivity of SLE cells remain under investigation.

Adaptation of human diploid fibroblasts in vitro to serum from different sources.

The growth of two human diploid skin fibroblast cell lines, originally grown in medium supplemented with foetal bovine serum and later adapted to medium supplemented with newborn bovine, bovine calf or horse serum, has been studied. Prolonged generation times increased cell volumes and decreased plating efficiencies were observed in cultures grown in newborn bovine, bovine calf or horse serum. In general, the deleterious effects were most severe as a result of growth in bovine calf or horse serum. In the light of the present findings, we believe investigators should exert great caution in switching human fibroblast cultures from foetal bovine serum to alternative sera, even at times of scarcity and high prices.