Environmental Wavelengths of Ultraviolet Light Induce Cytoskeletal Damage

Glen B. Zamansky, Ph.D. and Iih-Nan Chou, Ph.D. Department of Mi crobiology , Boston University School of Medicine, Boston, Massachusetts, U .S.A .

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 cel-lular fun ctions. The potential importance of ultraviolet light-indu ced damage to non-DNA targets has received little attention. Since the cy toskeleton is ;;. n im portant par-ticipant in the control of normal cell growth, the Hlicro-

Epidemiologic and ex perim ental evidence has esta b-lished solar ultravio let (UV) lig ht as the major ca use of skin cancer [1 ,2]. C hron ic ex posure to sunlig ht also damages derma l co nn ective tissue and contributes to the agin g of hum an skin [3,4]. A grea t deal of effort

has therefore been expend ed trying to elucidate th e m echanisms by which UV li ght alters no rmal cellul ar fun ctions. The majority of past investi gation s ha ve exa min ed DNA dam age resulting from exposure to short wavelength UV light . This has left many in-vestigators with th e impression th at pyrimidine dim ers are the sole lesion res ponsible for solar ca rcinogenes is. Humans are not exposed to such short wavelength UV lig ht, however, since vvavelengths below 290 nm do no t penetrate the atmospheric ozone layer. Furthermo re, th e transformation of norm al cells to a cancerOus phenotype appea rs to be a co mplex multistep process, ITlost o ften described as in volving stages of initiation and pro-ITlotion [5,6]. C urrent experim ental evidence suggests that ini-tia tion is ca used by geneti c dam age, and that promotion results fro m epigeneti c events. Since ex posure to promoters is not re-quired for tumor induction by "compl ete" carcinogens, most in ves ti ga tio ns into the ca rcinogeni c properties o f UV light con-tinue to emphas ize its genoto xic ca pability.

[s it likely th at UVB- (290-320 n111) o r UV A- (320-400 nm) induced alte rations of non-DNA targets ma y contribute to the d evelo pment of cellular dysfun ctio ns? It has been known for m an y yea rs tha t environmental waveleng ths of UV li ght induce bio-logicall y important lesions at non-DN A sites in bacterial cells [7].

Manuscript received December 15,1986; accepted for publication April 6, 1987.

T his research was supported in part by a grant from the Boston Uni-versity Communi ty Technology Foundation.

Heprint reques ts to: Glen B. Za mansky, Boston University School of M edicine, Department of Microbiology, 80 East Concord Street, Boston, MA 02 11 8

Abbreviations: HBSS: Hanks' balanced salt solution PB S: phosphate-buffered saline UV : ultra violet UVA: 320-400 11m UV light UVB: 290-320 11m UV light UV C: 200-290 nm UV light

fil aments and microtubules of UV irradiated human skin fibroblasts have been studied usin g fluorescence micros-copy. Polychromatic ultraviolet light, co mposed of envi-ronmentally relevant wavelengths, was found to disrupt the cytoplasmic microtubule complex in a dose dependent m anner. The indu ction of micro tubule disassembl y did not co rrelate with the cyto toxi city of ultraviolet light of vary-ing composition. J Ill ves t Deml(l/o/ 89:603- 606, 1987

Evidence has begun to accumul ate in m ammalian cell studies that les ions other th an pyrimidin e dimers m ay be in volved in the carcinogenic, mutageni c, and lethal effects of solar UV light [S-15]. Alth ough in ves tigato rs continue to seek additio nal DNA lesions to explain thesc findings, the importance of damage to other cellular components must also be considered.

The cyto plasm of eukaryo ti c cell s contains an intricate network of fi lamentous stru ctures th at arc co llectively referred to as the cytoskeleton. The three m ajor components of the cytoskeleton arc mi crotubules, mi cro fil am ents and interm edi ate fil aments /1 6). Since the cytos keleton is an important participant in the contro l o f normal cell g rowth , we have begun to explo re the possibility that UV li ght m ay induce cy toskeletal changes. We have been particularly interested in cytoskele tal alteratio ns induced by polychro m atic UVB and UV A li g ht sources, since they mo re closely simulate the UV lig ht to w hi ch we are routinel y exposed. Comparisons have also been m ade w ith the m o re commonl y investigated, 254 nm UV lig ht.

MATERIALS AND METHODS

Cells AG1522, o bta ined from the Institute for M ed ica l Resea rch (Ca mden, N ew J ersey), is a no rm al, dip loid hum an sk in fibrob las t cell strain . Cells were g rown in Eagle's minimal essential m edi um (G ibco, Grand Island , N ew Yo rk) supplemented w ith 10% fetal calf se rum , 0.9 gi l D-g lucose, 0.66 m g/l sodium pyru vate, 11 0 U / ml penicillin and 110 ug / ml streptom ycin sul fa te. C ultures were incubated at 37°C in :1n atm osphere of 95% air: 5% CO2,

UV Light General E lectric GST5 germicidal lam ps, Westing-house FS40 lamps, and Sylvania FR40T12 lamps were our sources of UVC, sun lamp, and UV A li g ht , respectively. T he UVC lamps emit grea ter th an 95% of their energy at 254 nm. The spectra of li ght transmitted by the Westinghouse FS40 and Sy l-vania FR40T12 lamps throug h po lysty rene culture dish covers have been published [1 7]. BrieRy, the sun lamps transmit ap-prox im ately equa l am o unts of li g ht in the UVB and UVA wave-bands , with a peak emissio n in the UVB reg ion between 310 and 315 nm . More than 9S% of th e li g ht fro m the UVA lamps, w hich emit nuximall y at 350- 355 nm, is in the UV A waveband . UVC dose rates were determin ed w ith an International Li ght (N ew burypo rt , M assachusetts) IL 254 germi cidal photometer. Sun lamp and UV A dose rates were determined w ith an lnter-

0022-202X/87/S03.50 Copyright © 1987 by The Society [or In ves tiga tive Dermatology, Inc.

603

604 ZAMANSKY AND C HOU

Figure 1. Microtubules (A,C,E,G) and microfi-laments (B,D,F, J-f) in AG1522 cell s. No UV light (A and 8); UV C, 100 J/m2 (C and D) ; s.un lamp, 5000 J/m2 (E and F); UVA , 100 kJ/m2 (G and H).

national Light I L 443 photometer. UVC cultures were irradiated without their co vers at a dose rate of approximately 0.4 J / m 2/sec. Sun lamp and UV A cu ltures were irradiated thro ug h their pol y-sty rene covers (0.8 mm thi ck) at dose rates of approximately 4.6 and 29.7 ] / m2/sec, respectively. In order to minimize variation of slln lamp or UV A lig ht transmiss ion through the plasri c, one lot of culture dish covers was used for all experim ents.

Cytoskeletons Eighteen hours afte r plating AG1522 cell s onto glass covers lips in 35 mm culture dishes (Falcon) , cultures were rinsed w ith Hanks' balan ced salt solution (HBSS) containing 15mM HEPES and UV irradiated in the presence ofHBSS. Unless stated otherwise, the cells were fix ed and cyroskeletons extracted im-med iately following irradiation. Fixation, ex traction and fluores-cent staining w ere perfo rmed as described r1 8J. BrieRy, imme-diately followin g irradiation, cu ltures w ere rin sed with PM2C buffer (0.1 M PIPES, 1 mM M gS04 , 2 111M EGTA and 2 M g lycerol, pH 6.9) and fixed in 3.7% form aldeh yde in PM2G buffer. C ul-tures were then washed w ith phosphate-buffered saline (PBS) , treated w ith 0.1 M g lycine and extracted with 0.3% Nonidet P-40. The fi xed cyroskelerons were in cubated wi th rabbit anti-tubulin antibodies to label the mi crotubul es and with the fluores cent re-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

agent NBD-phalla cidin to label th e mi erofilaments. Covers lips were then was hed w ith PB S and treated w ith goa t anti-rabbit ant ibodies conju gated to rhoda nlin e. In this m anller, micro tu-bu/cs and mi cro filalllcnts of the sa me cells were Iabellcd. Cover-slips were m ounted on glass slides prio r to exa minatio n in a Niko ll Ru o rescence mi croscopc. Using coded cove rsli ps to avoid COUnt-in g bias, the percent o f cell s w ith intact mi crotubu/cs and/or Illicrofi laments was determined by exa lllining 200 cel/s ill ran-do ml y selected field s at each UV dose. Cells were sco red as la ck-in g an intact micro tubule co mpl ex if mi cro tubules we re no t ob-served extending from an o rga nizin g center to the periph ery of the cell throughout th e cytoplasm . Photographs were t:lken at a m agnifi ca ti on level o f 125 x .

HES ULTS

We have investiga ted th e effe cts o f UV lig ht 0 11 cy roskclcta l mi-crotubules and mi crofibm cnts in normal, diploid human skin fibr oblasts. The mi crosco pic appea rance of mi cro tubul es and mi-cro filaments in control ;JJld irrad iated AG1522 cells is shown ill Fig lA-H. The progressively hi gher doses at lo nge r wavelen gth s o f lig ht were selected sin ce the efficiency of inducin g er ythema,

VOL. 89, NO.6 DECEMBER 1987 UV- INDUCED CYTOS KELETAL DAMAGE 605

Table I. Disruption of Microtubules by UVC, Sun Lamp o r UVA Lig ht"

uvc Sun Lamp UVA Dose U/ 1112) Intact MT (%) Dose U/ 1112) Intact MT (%) Dose (kJ/m2) Intact MT (%)

0 96.7 ± 0.3 0 95 .6 ± 1.2 0 95.7 ± 0.7 10 96.8 ± 1.0 750 94,3 ± 0,8 25 92.2 ± 3.1 20 96.3 ± 0.4 1500 90.8 ± 1. 4 50 80.4 ± 2.9 40 94.2 ± 1. 2 3000 61. 6 ± 12.8 75 68.0 ± 5. 1 75 95 .8 ± 1.7 5000 45.6 ± 8.5 100 44.2 ± 3.5

100 91. 5 ± 1.4 7500 27.5 ± 10.2 150 33.0 ± 6.7

UT he percent of cells w ith intact microtubulcs is presented :IS rhe l11ean ± I sran&ud e rror of 3-5 independent experim ents :H each UV dose. 200 Cells were exam ined in random ly selected microscopic fields in each cx pcrimcnr. Coded coverslips were used to :lvoid coullting bias. T he percent of I11 ock-irradi:ucd cel ls wi th intact microrubulcs was 91.3 :!: 1.9.

cellular in ac tivation , mutagenes is, transfo rmatio n, and o ther bi-o logic effects usuall y decreases as the wavelen gth of li g ht in-c r eases. As expected [1 6J, the microtubules of un irradiated AG1522 cells (Fig 1 A) appea r to eminate from perinuclea r mi crotubule organ izin g centers and extend throug ho ut the cy toplas m. The organ iza tion of microtubul es remains undisturbed in UVC -ir-r-adiated cell s (Fig 1 C) . Such a di stin ct network of micro tubules, however, is no t consisten tly o bserved in the cytoplas m of sun la mp- (Fig 1 E) o r UV A- (Fig 1 C) irradiated cells. Although th e microtubule orga nizin g center rem ains detectable, mu ch of the antitubu lin s tained cytoplasm takes o n a fine powdery appea rance in these cells. The extent to which this apparent disassembl y or fragmentation of microtubules occurs depends o n the UV dose and va ries from cell to cell. The mi crofilaments of the cells de-picted in Fig l A, C, E, and C are shown in Fig 1B, D, F, and H , respectiv ely. No changes were o bserved in the act in mi cro-filam ent bundles which stretch across the leng th of control and irradiated cells. As reported by others usin g NBD-phallac idin to s t a in mi crofil am ents [19], varying patterns of microfil am ent bun-dles were observed in AG1522 cells. These included cells with h eavy bundles, fin e bundles (e.g . , Fig 1 B), fine and heavy bundles (e. g., Fig 1 D ), o r no bundles (usually fewer than 5% of the cells).

In order to quantitate the disruption of microtubules, we per-formed dose res ponse experiments in which the percent of cells wi th intact cytoplasmic microtubule complexes was determined. As can be seen in Table I, intact micro tubules were found in cell s irradiated with UVC doses as high as 100 Jlm 2. Exposure to sun la mps resulted in the disruption of microtubul es, decreases in intact microtubules usuall y being observed after exposure to 3000 Jlm2 , Experiments with UV A light indica ted that it too causes a dose-dependent loss of organized micro tubules. As found above, no perceptible alterations of mi crofil aments occurred in UV C- , Sun lamp-, o r UV A-irradiated cells.

In all of the above experiments, cells were fi xed and cytoskel-e tons extrac ted immediately after exposure to UV light . In order to in ves ti ga te if time dependent changes occur in microtubules, we have also performed experiments in w hi ch cells were rein-cubated in g rowth m edium for vary ing lengths of tim e after irradiation (data not shown) . We have observed no increased level of d amage in cells exposed to UVC (5 or 10 J / m 2) or sun lamps (250 or 1500 J / m 2) as lon g as 24 h followin g irradiation. We have a lso observed no in creased accumulation of damage up to 7 h a fte r exposur-e to a sun lamp dose o f 7500 J / m 2. The detachment

Table II. UV Light Sensitivi'ty of AG1522 Cells"

uvc U/ m2) Su n lamp U/m 2) UVA (kJ / m2)

Do

2.3 ± 0.2 303.0 ± 12.3 47.2 ± 6.2

2.8 ± 1.0 484.8 ± 86.2

48.1 ± 8.2

8.2 ± 0.9 1182.6 ± 80.1

156.7 ± 12. 1

"D o, Dq, and 010 va lu es (mean:!: 1 standard error) were determined by usin g a I;near reg ression an alysis of the pooled data from 3-5 experiments for each source of UV light [141 .

of dying cells prevented th e in ves tiga ti on of longer times at this hig her dose.

U sing a standard colo ny forming assay, we have previously investiga ted the survival of AG 1522 and several o ther hum an sk in fibroblast cell s train s ex posed to UV lig ht [14J . Three param eters that describe the shape o f survival curves have been summ arized fo r AG1522 cells in Tab le II. T he Do va lu e is the dose required to redu ce surv ival to 37% in the expo nential po rtion of the curve an d is equi va lent to the in verse of th e slope of the st raight line portion of the curve. The D q va lue, determined by extra po lating the lin ear porti on of the curve to 100% survival, m eas ures the w idth of the shoulder and is indi ca ti ve of the cellular ability to accumulate sublethal dam age. The DIO va lue, the dose requ ired to redu ce surviva l to 10%, is a usefu l param eter, since it reflects the size of the sho ulder and the slo pe of the exponential decl ine. The data in Table II dem onstrate that th e disruptio n of micro-tubules occurs after ex posure to doses of sun lamp and UV A lig ht , w hi ch are significantl y less lethal th an 100 J / m2 UVC, a dose at which no microtubul e damage is observed . In fact, ex-trapolation of the UV C survival data indicates that onl y one out of approximately 1016 AG1522 cells survive exposure to 100J/ m 2; whereas ex posure to the microtubule-dam aging dose of l 00 kJlm 2

UV A results in the su rviva l of g rea ter than 15 o rders of magnjtude m ore cell s. Thus, the induction of mi crotubul e damage does not appear to correlate with the cy totox icity of UV light of varying wavelength. It w ill be of interest to determine if this lack of correlation also occurs in' cells that are hyperscnsiti ve to UV li ght, o r if the disruption of microtubules m ay contribute to their hypersensitivity.

DISC USSION

The current stud y represents the first dem o nstratio n of UV in-duced disruption of cytos keleta l elements in cultured cells. Jim bow and associates o bse rved a r-cdistriblltio ll of intact mi crofi lam ents and microtubules from the perinucl ear regions to t he periphery of the cytoplasm and the dendritic processes of m elanocy tes that had been exposed in vivo to UV A and visible light [20]. It was suggested that this redis tribution plays an impo rtant role in the transport o f melanosomes during the immediate tannin g reaction. Honigs m ann and colleagues, howevcr, recentl y reported th at chemi ca ll y induced disassemqly of micro tubllles o r mi crofila-m ents does not inhibit th e immedi ate tannin g reaction followin g UVA irradiation of explanted skin specimens [21J. Zaremba and cowo rkers have shown th at UV C li ght inhibits the in vitro po-lym eri za ti on of tubulin dimers into no rm al mi cro tubular struc-tures [22J . Exposure to 280 nm UV lig ht was approximately tw ice more efficient than 254 nm and eight times more efficient than 300 nm light in causing this inhibitio n oftllbulin asse mbl y. From the data ofZarel11 ba and coworkers, it can be ascertained that the dose of 254nl11 UV light required to reduce tubulin polymeri-za tion by 50% would be approximately 3000 Jlm2. T hough po-tentiall y useful for in ves tiga ti ons into the pol y m erization of mi-crotubules in vitro , our data clearly indi ca te that such an exposure to cultured cells would no t be biologica ll y m eaning ful , sin ce a dose 30- fo ld lower is already extraordin aril y toxic.

606 ZA M ANS KY AND C H OU

'The indi vidual co mponents orthe cy toskeleton are stru cturall y associated w ith each oth er as well as w ith th e cellul ar m embrane and nu clear matri x 1'23-26]. It ha s therefore been sugges ted that the cytoskel eton ma y serve as a criti ca l means of transmitting externa l sig nals to th e nucleus. The co mplex network of cy to-skeletal stru ctures also participates in the regulation of cell growth, shape, and motility , the spatial arrangem ent of o rganell es , and secretory processes 1'1 6,23-26]. It is thu s reasonable to expect th at UV li g ht- induced perturbatio ns of the no rmal assemb lage of th e cytos keleton co uld result in a variety of functi onal conseq uences. Indeed , cy toskeleta l ab no rm aliti es have no w been associated w ith seve ral patho logic phenomena, including th e malignant transfor-mation of cells [27,28]. It is also intri guin g to note that tumor promoters have recentl y been fo und to ca use stru ctural chan ges in th e three major cytoskeleta l co mponents [29-33] .

We believe th at our studi es provide an impo rtant, new approach for stud y ing cellu lar damage induced by solar wavelengths of UV li ght. Our data demonstrate that exposure to polychromatic UVB and UV A li g ht dama ges cytoskeletalmi crotubules. The UV doses at whi~h we obse rved disruption of th e mi cro tubul es are well within the ran ge of environmenta l UV ex pos ures [34,35), though our UV lamps do not reproduce exactl y the soJar UV li ght spec-trum. T his stud y assul11es an added impo rtance, since light sources similar to those in o ur experiments are al so used in the trea tm ent of va rioLls ski n diseases [36]. Furthermore, ex posure to very hi gh doses from such UV la m ps fo r purel y cos nl.etic reasons has be-cO l11 e mo re C0 l111110n as th e popularity of tanning parlors has increased.

We wish 10 express all" appreciaf ioll fa g radllofe sflldellfs J.P. Shaw alld B. Perrillo fo r fh ei,· flICH/g hfjid co llfribllfiolls fa fhis sflldy.

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TH E JOU RNAL OF INVESTIGATIVE DERMATOLOGY

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