AJEBAK 56 (Pt. :^) 287-296 (1978)
CHROMOSOME DAMAGE AND DNA REPAIR INDUCED IN HUMAN FIBROBLASTS BY UV AND CHOLESTEROL OXIDE
hy P. C. PARSONS A.ND PATRICIA GOSS (From the Quci'iisland Institute ni Medical Research, Herstoti, Queensland, Australia.)
(Accepted for pitblication December 19, 1977.)
Sumniar>\ In luiniaii lilirolblasts. cholesU-rol oxide induced a similar degree of chrnnuwome damage (H-(i% of inetaplia.st's) and DNA repair synthesis (8-10^ of cells with lijjlitl>-lai)i'llL'd nuclei) iw low dtwes of ultraviolet litilit (UV), but did not produLc single-stratid DN.A breaks or UiN.A diuinijic detectable hy inhibition of th\inidiiie incnrpuratiini. f^hroniosoiiie aberrations were deteeted up to S week.s iifter treatment with cholesterol oxide and VW Cined treatments had almost additixe ellect.s ini the frequency iif chrotnosonie aberrations liut mit im repair s\ntht'sis. Multiple daily doses of UV did not came more atx'rrations than a single dose, Attempts to transfiirn] two fibroblast strains from nfirinal donors and three derived from nielaiiotna patients using siiiHlc atid combined treatments of U\', cholesterol oxide and hypertherniia ( !()°) were unsiicce.ssful.
IXTRODICTION. The strong circumstantial evidence for a role tjf solar-derived UV in the aetiolog\- of huniati .skin cancer (Urhacli, 1975) aticl the ability of UV to trans(ortn cultured rodent cells (CMian and Little. 1976; DiPaolo and Donovan. 1976; Mntidal atld Ili'idelherger, 1976) enconrages attetnpts to tran.storin normal human cells by UV in vitro. Au expcrinietital model of this type wt)uld be vahtabK' for testing hypotheses coticerning the iiidttction and prevention of skin Itiiiiottrs in man. Thai snili a s\stein ha.s tiot yi't l)een reported ina\' be due to the more efficient DNA excision repair systetu it) human cells or a requirement for promoting factors sueh as chemical.s, viruses or other solar \va\elengths (Frectiiiui atid Kiit)x, 1965; Goss and Parsons. 1976; Mondal and Heidelln-rger, 1976). Alternatively, if UV should act indirectly through the in situ Formation of a chemical carcinogen, then tratisformation tnight best be eflFected b\' direct administration of the coinponnd. Gholesterol-a-oxide, for example, would appear
P. G. PARSONS .AND PATRIGL\ GOSS
28S
to be a candidate as it is carcinogonic iti rodents and can be generated iu humau skin by V\ oxidation ol eholesterol ( Blaek and Lo, 1971). approx. 80 mg of which is excreted daily tinough the .skin (IJhattacharyya, Cotinor and Spcctor, 1972). Oiificnlties experienced in the transformation of human cells with physical and chemical agents have led to studies t)f temporary effects such as chromosome damage and DNA ri-pair synthesis wliieh tnay well be part of the neoplastic process (Stich et al, 1975). Chromosome alM'rrations were considered a less specific indicator ihati DNA repair s\nthesis bitt useful for thi* stttd\' of longterm effects (Al)l)()ndatH)Io. 1977; Stieh cl al, 1975). Contititted inliibition of semi-conservative DNA synihesis following removal of the test compomid is an attracti\'(' assay of DNA damage by careitiogeiis, although not x'cry s('tisiti\e for alkylation dattiage (Painter, 1977). We liave atteniptc'd to triuisforin nortnal and tnelanoma-derived humau fibroblasts using UV and cholesterol oxide, both alotie and in eombitiation with h\pertherinia. In addition to tnotutoring for transformed loci, the target cells were tested tor chrotuosotne atid DNA damage to assess the carcinogenic potential of cholesterol oxide in human cells and to obtain information which could guide future transformation studies. MATEIU.ALS AND METHODS.
Riohfiica} matrricils The orijiiiis of the human [ibnibkst striiiiis are jjiven in Talile I. The fibroblasts (nun melanoma patients nscnibled normal librnblusts with rt'Spect to mnrphK>'. contactinhibited growth and inability tii grow in ayar. The hunmn melanoma cell lines MM96 and TABLE 1. Origin of hunian fibrohlast strains used as target cells. Age
Sex
Biopsy site
17 32
M F M
Anterior forearm .Anterior forearm Anterior furearm
Melanoma MM185-F MM288-F
72 54
F F
MM?3!-F
R5
F
Eye Inguinal lymph mule metastasis Ingtjinal tymph mule metastasis
Strain No mini BB
SGB DJM
21
MM253 ha\e been deserilH-d (Coss and Parsons. 1977). Celk were cultured in RPMI 1640 medium containing 16? foetal calf serum and periodically assayed for MycopltVima (Goss iind Parsons. 1976). Cell sur\i\als were determined b> colony fiirmation on a plastic surface as previdusK descrilKti (Goss and Parsons. 1977). Fur experiments \\s\\\)i high o\yji.'ii levels, tells wi'rt' Kni«ii in plastic Ha.slis which were Hushed with th? requiretl gas mixture (o.\ygen plus nitrogen) and .sealetl. Cholesterol-a-o.vide was prepared from chriimatographic grade cholesternl b\ thv ni?thod of FiestT and r'irKcr (1007).
CHOLESTEROL OXIDE AND HUMAN CELLS
289
Attempted transformation Fihroblasts (approx. 3 x lO'' per 60 mm dish or 10" per 80 mm dish) were irradiated (Goss and Parsons. 1976) or treated with cholesterol oxide (1 mg/ml solution in acetone) at the lc\cl.s stilted. In combined treatments, cholesterol oxide was added immediately following irradiation. Treatments at 40° were carried out in a CO^ incubator during the first 4 h of treatment with cholcstcrd! oxide. In most experiments control cultures contained chok'sterol at the same concentration as cholesterol oxide. All culture.s were trypsinized when confluent and contimied in passage for the periods indicated, Chromasonw amihj.sis Cells were i)l)tained from cultures beinji passaged for the transformation assays and nietaphase spreads were prepared aceorfling to the inethod of Moorhead and Nowell (1964). The precautions in scoring aberrations recommended by Cohen and Hirschorn (1971) were observed. DNA daninae and DNA repair synthesis was determined by autoradiography as previously reported (Coss and Parsons. 1976). Detection of single-strand breaks by sedimentation of nucleoids was essentially the method of Cook and Brazell (1976). Approximately 5 x 10^' cells, harve.sted by lr>psinizati()n at 36" for 5 min, were su.spended in 50 ul of phosphate-buffered saline (pM 7-2) and gently mixed with 150 [i\ of lysing snintion (0-67'i' Triton X-100. 2-6 M NaCl, 2-6 mM EDTA, 0-13 M Tris, pH 8'0). After 1.5 min the mixture was gently transferred using a wide-bore plastic tip to a 12 ml (9 cm) gradient of 10-30% sucrose ejmtaiiiing 1'95 M NaCl, 1 mM EDTA and O-Ol M Tris (pH 8-0) and spim at 16,000 rev./niin (37.000 x g) in an International 488 rotor at 20° for 20 min. Gradients were fractionated by siphoning through an LKB Uvicord II monitor operating at 260 nm. In tracer experiments, apprnx. 10' cells grown in 30 mm diameter dishes were prelalielled for 18 h with [»i^//iy?--^H]-thyniidine (2 HCi/nil) or [2-i-'C]-thymidine (0-5 ^Ci/ml). the gradients being fractionated by needle puncture followed by trichloroacetic acid precipitation and eoUectiim on gla,ss fibre discs. Cultures to be analysed by alkaline sucrose sedimentation (Coss and Parsons. 1976) were labelled siuiilarly. The method of Painter (1977) was used to studj' the cfFect of UV and cholesterol oxide on thymidine uptake in MM253 cells. Triplicate cultures prelabelled with ^'C-thymidine as above were pulsed with ^iH-thymidine (10 (iCi/nil for 15 min) at hourly intervals following treatment with UV or cholesterol oxide. The ^H/i-iC ratio was compared with the ratio from eontrol cultures har\'ested at the .same time.
RESULTS. Plating efficiencies at hi^h oxygen levels Eollowing the tiiethod of Mitchell, Elgas and Balk (1976) for the selective growth of transformed chick cells, 2 human fibroblast strains and the melanoma lino MM96 were used as a modt-l system to determine whether high oxygen levels would select for tlie growth of transformed human cells compared with normal cells. A difference in plating efficiency was oh.served {Fig. 1), but was too small to be used as a selection method. Attempted transformation The dose-survival respon.sc of several fibroblast strains vi'as determitied to allow cells to be irradiated at known survival levels (Fig. 2). In agreement with the results of Lehmann et a]. (1977), there was no significant difference in sitrvival between the nortnal and melanoma-derived strains. Post-incubation at 40' did not greatly reduce survival, and cholesterol oxide had no cytotoxic effect iu the conceutration range 0-5-10 jLtg/ml (results not shown).
290
P. G. PARSONS AN-D PATRICIA GOSS T
100 -
10 •
_
1
1
20
40
60
80
Percent Oxygen Fig. 1. Effect of tixygen level on tx-Il surviviil expressed as perci'ntage of plating efTic/ifncy in air: O O- MM9e; • Q, BB: A A- MM331-F. Bars ± standard deviation of 6 replicates.
100
10
20
Fluence (ergs x mm" -) Fig. 2. Effect of hyperthermia on UV survival of fibroblasts: O - - - O- MM283-F: • - • - • , MM280-F with 4 h post-incubatioLi at 40°: D D. SGR; • • . SGB with 4 h post-incubation at JO". Bars ±: standard dc\iatioii nf 6 rt'plicates. Not shown when less than twice the .symbol size.
CHOLESTEROL OXIDE AND HUMAN CELLS
291
In one transformation attempt DJM fibroblasts were treated with UV (15 ergs X mm"-), ehole.sterol oxide (10 fig/m\ for 24 h) heat {40' for 4 h) or combinations of these agents. After 1 week the treatments were repeated and the cultures then maintained for 38 weeks. During the second half of this period both control and treated cells heeatne senescent without any evidence of transformation as judged by morphology or multi-layered foci different from controls. The other fibroblast strains were given 5 daily treatments and observed for periods of 4-6 weeks, again with negative results. Chromosome analysis Cultures from transformation experiments in which a UV Huence of 2 ergs X mm~- was used resumed uoruial growth rate after 1 week and were therefore considered suitable for chromosome analysis. Approx. 4-20^ of cells were in metaphase. Most of the abnormalities ( > 95%) were cliromatid breaks or deletions, with a similar average in al! experiments of approx. 1-3 aberrations per scored metaphase. The aberration frequencies resulting from single treatments of 1 normal (1 experiment) and 1 inelanoma-derived fibroblast strain (2 experiments) are sliown in Table 2. All probabilities were calculated on the totals for both strains using the chi-s(iuared test with continuity correction; similar probabilities were obtained by eomparitig means of the weekly values using the t-test. Comparison of results obtained at a single time point usually gave a significant difference, e.^., 1 week after treatment with cholesterol oxide, p < 002 compared with the eontrol. Based on the signifieance levels in Table 2, aberrations were iudueed by cholesterol oxide alone and enhanced by hypcrthermia when cholesterol oxide and UV were used separately and together. Cliolesterol oxide and UV-indueed aberrations were still present after 8 weeks (p < 0()5). Target cultures subjected also to hypertlicrmia initially showed a high aberration frequency which decreased over the 8 week period (p < ()-05). The aberration level iu untreated cells, the number of metaphases counted and the lifespan of the eell strain limit the period over which observations can be made. Thus, Marshall and Scott (1976) found that aberrations induced in normal human fibroblasts by UV (5 ergs X mm--) returned to control levels (7'/) after only 3 days. Most types of treatments gave approx. twice the aberration frequency in the melanomaderived strain as in the normal strain. The relative significance of donor age or melanoma origin conld not be assessed because of the limited number of strains studied. Aberratiou frequencies were also increased by multiple UV heatment of SCB (Table 3), MM185-F and MM288>F fibroblasts (re.sults not shown), but were not significantly increased compared with a single treatment (p > 0-1). DNA repair synthesis For determination of DNA repair synthesis, as judged by sparsely-labelled nuclei iu antoradiographs, separate fibroblast ciiltiucs were treated at higher levels of eholesterol oxide and UV than used iu tlie transformation experiments
292
P. C. PARSONS AND PATRICIA COSS
o c OO
DDO^iOO
o o VV
V V V V VV
( S "*•
293
CHOLESTEROL OXIDE AND HUMAN CELLS TABLE 3. Chromosome aherrcitions in SGB ftbrohlasts jollowiit^ 4 daily treatments. Weeks after treatment
Treatmenf^' Control
1
2
3
3t
-1
2
Total
%
10
2-5
9
17
9-3§
4
21
5-3
8
W
10
7
40
6
7
UV + 40
14
7
10
8
39
9 8§
UV ^ 40 + c o t
20
M
13
6
?6
10 0§
11
treatment levels as Table 2. fNumbers of metaphases with aberrations out of lOO metaphases examined. tOnly one treatment with cholesterol oxide. SSignificance compared with control, p < 0 001.
and liaivt'sted 4 h later. U\' increased the proportion of sparsely-lubelled nuclei (Tahle 4), showing that the method was effective in detecting repair .synthesis. Cholesterol oxide., bnt nt)t cholesterol, was also fonnd to indnee repair synthesis. The efFeet was almost additive with VV in tlie SCB and MM288-F strains (Jitter results not shown) but not in the MM331-F strain. Post-incnbation at 40~ had little effect. TABLE 4. Iinliiclioii of DNA repair synthesis in human fihnihlasts hy cholesterol oxide and UV.
Treatment"
Per cent sparsely-labelled nucleif MM331-F SGB
Control
1-4 It 0-3
1-6 ± 0-28
40
1-9 ± 1-5
2-3 ±
1-8
CO
8-5 ± 0-42
10-6 ±
2-3
CO + 40
10-'* + l)-7
10 0 ± 0-6
UV
8-1 ± 0-4
10-5 ±
3-2
UV + 40
8-9 ± 0-7
11-5 ±
4-4
CO + UV
15-4 + I'4
CO 4 UV + 40
14-6 ±
11
•^Control. 4 b intubation at 3ft" with H) iig/ml cholesterol; 40. 4 h incubation at 40° CO, 4 h incubation with 10 fig/ml cholesterol oxide: UV. 10 ergs x m m - - . fAverage of duplicate slides ± standard deviation (500 cells counted per slide). Significance compared witb control, p < 0 0 1 (except 40° treatment).
294
P. G. PARSONS AND PATRICIA GOSS
Detection of single-strand breaks The potential of alkaline sucrose gradients to resolve single-stranded DNA was improved using a mixture of -^H and "G-Iabelled test and control cells in the same gradient as recotiiniendcd by Dingmaii and Kakunaga (1976). The DNA profiles of strains SGB and M M ' 3 3 1 - F treated with cholesterol oxide for 24 h showed no difference from controls; no cotnbined treatments were used. Tlie same result was obtained when tlie cells were conversely labelled. Using the more sensitive nucleoid sedimentation method, material from UV-irradiated SGB cells (60 ergs x m m - - ) showed greatly rednced sedimentation compared with untreated cells (Fig. 3 ) : in other experiments a dijfference was distinguishable at 10 ergs x turn--, in agreement with Cook and Brazell (1976). Gholestcrol oxide, however, showed no .significant effect in repeated experiments using the SGB and BB strains (Fig. 3), This was also the case when mixtures of labelled cells were analysed in the same gradient.
O
D
0.6
04
0.2
Distance Sedimented Fig. 3. Detection of DNA singlc-strantled breaks in SGB fibrnhlasts by sedimentation of nucleoids (sample zone not sllov^^l). A, imtreittecl cells. B, L h after UV irradiation (60 ergs X m m - - ) . C. 24 h after addition ot cliolesteriil oxide (10 jig/ml), D, O O. untreated cells; • • , 24 ll after addition of cholesterol oxide (10 [ig/ml).
Detection of DNA daimige by inhibition of thymidine
uptake
The melanoma line MM253 was used for tliis test because of its sensitivity to UV (Lavin, Good and C^oitnsilman, 1977) and alkylating agctits (Goss and Parsons, 1977). atid its enhanced ability to incorporate labelled thymidine. A UV dose of 50 ergs x nini-= gave a 90^: decrease in DNA synthesis over 3 h, similar to the response ot HeLa cells reported by Painter (1977); 10 ergs x
CHOLESTEROL OXIDE AND HUMAN CELLS
295
produced a 30% decrease. A 1 h pre-treatment with cholesterol or cholesterol oxide (10 /ig/ml), however, had no significant e£Fect on thymidine ineorporation during this period. DISCUSSION. The present approach to UV transformation was planned as an attempt to circumvent the UV protection systems of human fibroblasts by various means: multiple doses, combined doses with a carcinogenic photoproduct, hyperthermia as a potential promotor, and use of target cells from aged melanoma patients. The lack of sneeess .so far may be due to a requirement for more specific inhibition of repair, or for synergistic factors as yet unknown. The choice of UV wavelength or target cell type should not be limiting factors, since sarcomas are readily induced in mice at this wavelength, the lack of sun-related sarcomas in man being explicable in terms oH thieker skin protection (Blum, 1959). However, closer simulation of the in vivo situation would be desirable, even if less convenient experimentally. Future attempts to transform hnman cells by UV conld be guided by recent experience with rodent cells where use of special contact-inhibited strains, use of phorbol ester (Mondal and Heidelberger, 1976), pretreatment with X-rays (DiPaolo and Donovan. 19761 and critical timing of treatments (Chan and Little, 1976; Mondal and Heidelbergcr, 1976) have led to success in vitro. Appropriate selection methods such as the agar cloning procedure nscd for viral transformation assays may be needed to detect what is evidently a very low transformation frequency. Several new observations, relevant to UV transformation of human cells, were made using carcinogen assays. The induction of chromosome abnormalities and repair synthesi.s by cholesterol oxide provided the first evidence for this compound having a careinogen-Iike effect on human cells. However, more detailed studies of the action of cholesterol oxide may be difficult because of the lack of detectable DNA breaks or other DNA damage. Although the process of UV carcinogenesis may presently be explained in general terms without recourse to an intermediary carcinogen, these results show that cholesterol oxide and other skin photoproducts should be further evaluated as potential carcinogens m man. The synergistic eflFect of hyperthermia on chromosome aberrations supports previous evidence (Goss and Parsons, 1976; Freeman and Knox, 1965) that increased skin temperature may play a role in UV carcinogenesis. The observation that some damage from both cholesterol oxide and UV remained at 8 weeks suggests that factors synergistic to the later stages of carcinogenesis could be effective long after UV exposure. Epidemiological studies of such factors would need to consider this possibility. Acknowledgements. The co-operation of Dr. J. H. Littk- and Dr. N. C. Davis, of the Quffiisland Melanoma Project, Princess Alexandra Hospital. Brisbane, is gratefully acknowledged. We wi.sh to thank Leanne Morrison and Juanita Yuen for excellent technical assistance. This work was supported by the Queensland State Government, and assisted by a grant from the National Health and Medical Research Council, Canberra.
296
P. C. PARSONS AND PATRICIA GOSS
REFERENCES. ABHONDANDOLO, A. (1977): 'Pro.spccts for CKJSS, P.. and PARSONS. P. G. (1977): evaluatinjf genetic damage in mammalian 'The efFect of hyperthermia and melcells in culture.' Mtitat. Res.. 42. 279. pbalan ou survival of human fibroblast strains and melanoma cell lines.' Cancer BHATTACHARYYA, A. K., CONNOR, W . E.. Res., 37. 152. and SPECTOR, A, A. (1972): 'Excretion LAVIN, M. F . , GOOD. M., and CO"NSILof sterols from ths skin of normal and MAN. C. (1977): 'Resistance of human hypercholcsterolemic humans.' J. Clin. melanoma cells to ultraviolet and ionizInvest., 51, 2060. ing radiation.' Proc. Au.st. Biochem. Soc, BLACK. H . S., and Lo. W. (1971): Torma10, 77. tion of a carcinogen in human skin LEHMANN, A. R.. KIKK-BELL. S., ABLETT, irradiated with ultraviolet light' Nature. C. F., HARCOURT. S. A., DE WEERD234, .306. K.'vsTEi.EiN. E. A.. KEIJZER, W . . and BLUM, 11. F. (1959): "Carcinogene.sis by HALL-SMITH. P, (1977 ): 'Repair of ultraUltraviolet Light", Princeton University \iolet liglil damage in a variety of human Press. Princeton. fibroblast cell strains.' Cancer Res., 37, 904. CHAN. G . L.. and LITTLE, J. B. (1976): MARSHALL. R. R.. and SCOTT. D . (1976): 'Induction of oncogenic transformation 'The relationship between chromosome in vitro by ultraviolet light." Nature. damage and cell killing in UV-irradiated 264, 442. normal and xeroderma pigmentosum cells.' CouKN, M. M.. and IIIHSCHOHN. K. (1971): Mtitat, Res.. 36, ,397. 'Cytogenetic studies iu animals.' In MITCHELL, R, S.. FLGAS, R. J., and BALK, "Chemical Mntagens. Principles and S. D. (1976); 'ProliferaHon of Rous Method.s for Their Detection", A. Holsarcoma virus-infected, but not of Iaender (editor). Plenum Press. New nonnal. chicken fibroblasts in oxygenYork, \'ol. 2, p. 515. em iched environment; preliminary reCOOK, P. R., and BRAZELL, I, A. (1976): port.' Proc. Nat. Acad. Sci. (U.S.A.). 73, 'Detection and repair of single-strand 1265. breaks in nuclear DNA.' Nature. 263. MoxDAL, S.. and HEIDELBERGER. C, (1976): 679. 'Transformation of C3H/10T1/2CL8 DiNGMAN. C. W., and KAKUNACA. T . nionse embryo fibroblasts by ultraviolet (1976): 'DNA strand breaking and reirradiation and a phorbol ester.' Nature. joining in response to ultraviolet light 260. 710. in nonnal human and xeroderma pigMooRHKAD. P. S., and NOWELL, P. C, nientosum cells.' Int. ]. Radicit. Biol. 30. (1964); 'Chromosome cytology." In 55. "Methods in Medical Research'", H. N. DIPAOLO, J. A., and DONOVAN, P. J. Eisen (editor). Year Book Medical Pub( 1976): 'In vitro morphologic translishers, Chicago, Vol. 10, p. 310. formation of Syrian hamster eells by PAINTER, R. B. (1977): 'Rapid test to l^V-irradiation is enhanced by Xdetect agents that damage human DNA." irradiation and imadected by chemical Nutiire. 205. 650. carcinogens.* Int. ]. Radiat. Biol, 30, 41. STICH. H . F . , LAM, P., Lo. L. W., KOROFiESKR, L, F., and FU:SEH. M . (1967): PATNTCK. D. J., and SAN, R. II. C. "Reagents for Organic Synthesis". Vol. 1. (1975): 'The search for relevant shortJ. Wiley and Son.s. New Y'ork, p. 136. term hioassays for chemical carcinogens: FREEMAN, R. G., and KNOX. J. M. (1965): th? tribulations of a modern Sis\'phus.' The factor of temperature in ultraviolet Can, } . Gcnt't, Cijiol. 17. 471. injury.' Arch. Environ. Health. 11, 477. UitHACH, F. (1975): 'Ultraviolet radiation: Goss, P., and PARSONS, P. G. (1976); interaction with biological molecules.' In 'Temperature-sensitive DNA repair of "Cancer, a comprehensive treatise"'. F. ultraviolet damage in hujuan cell lines.' F. Becker (editor). Plenum Press, New Inf. /. Cancer, 17. 296. York, Vol. 1. p. 441.
CHROMOSOME DAMAGE AND DNA REPAIR INDUCED IN HUMAN FIBROBLASTS BY UV AND CHOLESTEROL OXIDE
hy P. C. PARSONS A.ND PATRICIA GOSS (From the Quci'iisland Institute ni Medical Research, Herstoti, Queensland, Australia.)
(Accepted for pitblication December 19, 1977.)
Sumniar>\ In luiniaii lilirolblasts. cholesU-rol oxide induced a similar degree of chrnnuwome damage (H-(i% of inetaplia.st's) and DNA repair synthesis (8-10^ of cells with lijjlitl>-lai)i'llL'd nuclei) iw low dtwes of ultraviolet litilit (UV), but did not produLc single-stratid DN.A breaks or UiN.A diuinijic detectable hy inhibition of th\inidiiie incnrpuratiini. f^hroniosoiiie aberrations were deteeted up to S week.s iifter treatment with cholesterol oxide and VW Cined treatments had almost additixe ellect.s ini the frequency iif chrotnosonie aberrations liut mit im repair s\ntht'sis. Multiple daily doses of UV did not came more atx'rrations than a single dose, Attempts to transfiirn] two fibroblast strains from nfirinal donors and three derived from nielaiiotna patients using siiiHlc atid combined treatments of U\', cholesterol oxide and hypertherniia ( !()°) were unsiicce.ssful.
IXTRODICTION. The strong circumstantial evidence for a role tjf solar-derived UV in the aetiolog\- of huniati .skin cancer (Urhacli, 1975) aticl the ability of UV to trans(ortn cultured rodent cells (CMian and Little. 1976; DiPaolo and Donovan. 1976; Mntidal atld Ili'idelherger, 1976) enconrages attetnpts to tran.storin normal human cells by UV in vitro. Au expcrinietital model of this type wt)uld be vahtabK' for testing hypotheses coticerning the iiidttction and prevention of skin Itiiiiottrs in man. Thai snili a s\stein ha.s tiot yi't l)een reported ina\' be due to the more efficient DNA excision repair systetu it) human cells or a requirement for promoting factors sueh as chemical.s, viruses or other solar \va\elengths (Frectiiiui atid Kiit)x, 1965; Goss and Parsons. 1976; Mondal and Heidelln-rger, 1976). Alternatively, if UV should act indirectly through the in situ Formation of a chemical carcinogen, then tratisformation tnight best be eflFected b\' direct administration of the coinponnd. Gholesterol-a-oxide, for example, would appear
P. G. PARSONS .AND PATRIGL\ GOSS
28S
to be a candidate as it is carcinogonic iti rodents and can be generated iu humau skin by V\ oxidation ol eholesterol ( Blaek and Lo, 1971). approx. 80 mg of which is excreted daily tinough the .skin (IJhattacharyya, Cotinor and Spcctor, 1972). Oiificnlties experienced in the transformation of human cells with physical and chemical agents have led to studies t)f temporary effects such as chromosome damage and DNA ri-pair synthesis wliieh tnay well be part of the neoplastic process (Stich et al, 1975). Chromosome alM'rrations were considered a less specific indicator ihati DNA repair s\nthesis bitt useful for thi* stttd\' of longterm effects (Al)l)()ndatH)Io. 1977; Stieh cl al, 1975). Contititted inliibition of semi-conservative DNA synihesis following removal of the test compomid is an attracti\'(' assay of DNA damage by careitiogeiis, although not x'cry s('tisiti\e for alkylation dattiage (Painter, 1977). We liave atteniptc'd to triuisforin nortnal and tnelanoma-derived humau fibroblasts using UV and cholesterol oxide, both alotie and in eombitiation with h\pertherinia. In addition to tnotutoring for transformed loci, the target cells were tested tor chrotuosotne atid DNA damage to assess the carcinogenic potential of cholesterol oxide in human cells and to obtain information which could guide future transformation studies. MATEIU.ALS AND METHODS.
Riohfiica} matrricils The orijiiiis of the human [ibnibkst striiiiis are jjiven in Talile I. The fibroblasts (nun melanoma patients nscnibled normal librnblusts with rt'Spect to mnrphK>'. contactinhibited growth and inability tii grow in ayar. The hunmn melanoma cell lines MM96 and TABLE 1. Origin of hunian fibrohlast strains used as target cells. Age
Sex
Biopsy site
17 32
M F M
Anterior forearm .Anterior forearm Anterior furearm
Melanoma MM185-F MM288-F
72 54
F F
MM?3!-F
R5
F
Eye Inguinal lymph mule metastasis Ingtjinal tymph mule metastasis
Strain No mini BB
SGB DJM
21
MM253 ha\e been deserilH-d (Coss and Parsons. 1977). Celk were cultured in RPMI 1640 medium containing 16? foetal calf serum and periodically assayed for MycopltVima (Goss iind Parsons. 1976). Cell sur\i\als were determined b> colony fiirmation on a plastic surface as previdusK descrilKti (Goss and Parsons. 1977). Fur experiments \\s\\\)i high o\yji.'ii levels, tells wi'rt' Kni«ii in plastic Ha.slis which were Hushed with th? requiretl gas mixture (o.\ygen plus nitrogen) and .sealetl. Cholesterol-a-o.vide was prepared from chriimatographic grade cholesternl b\ thv ni?thod of FiestT and r'irKcr (1007).
CHOLESTEROL OXIDE AND HUMAN CELLS
289
Attempted transformation Fihroblasts (approx. 3 x lO'' per 60 mm dish or 10" per 80 mm dish) were irradiated (Goss and Parsons. 1976) or treated with cholesterol oxide (1 mg/ml solution in acetone) at the lc\cl.s stilted. In combined treatments, cholesterol oxide was added immediately following irradiation. Treatments at 40° were carried out in a CO^ incubator during the first 4 h of treatment with cholcstcrd! oxide. In most experiments control cultures contained chok'sterol at the same concentration as cholesterol oxide. All culture.s were trypsinized when confluent and contimied in passage for the periods indicated, Chromasonw amihj.sis Cells were i)l)tained from cultures beinji passaged for the transformation assays and nietaphase spreads were prepared aceorfling to the inethod of Moorhead and Nowell (1964). The precautions in scoring aberrations recommended by Cohen and Hirschorn (1971) were observed. DNA daninae and DNA repair synthesis was determined by autoradiography as previously reported (Coss and Parsons. 1976). Detection of single-strand breaks by sedimentation of nucleoids was essentially the method of Cook and Brazell (1976). Approximately 5 x 10^' cells, harve.sted by lr>psinizati()n at 36" for 5 min, were su.spended in 50 ul of phosphate-buffered saline (pM 7-2) and gently mixed with 150 [i\ of lysing snintion (0-67'i' Triton X-100. 2-6 M NaCl, 2-6 mM EDTA, 0-13 M Tris, pH 8'0). After 1.5 min the mixture was gently transferred using a wide-bore plastic tip to a 12 ml (9 cm) gradient of 10-30% sucrose ejmtaiiiing 1'95 M NaCl, 1 mM EDTA and O-Ol M Tris (pH 8-0) and spim at 16,000 rev./niin (37.000 x g) in an International 488 rotor at 20° for 20 min. Gradients were fractionated by siphoning through an LKB Uvicord II monitor operating at 260 nm. In tracer experiments, apprnx. 10' cells grown in 30 mm diameter dishes were prelalielled for 18 h with [»i^//iy?--^H]-thyniidine (2 HCi/nil) or [2-i-'C]-thymidine (0-5 ^Ci/ml). the gradients being fractionated by needle puncture followed by trichloroacetic acid precipitation and eoUectiim on gla,ss fibre discs. Cultures to be analysed by alkaline sucrose sedimentation (Coss and Parsons. 1976) were labelled siuiilarly. The method of Painter (1977) was used to studj' the cfFect of UV and cholesterol oxide on thymidine uptake in MM253 cells. Triplicate cultures prelabelled with ^'C-thymidine as above were pulsed with ^iH-thymidine (10 (iCi/nil for 15 min) at hourly intervals following treatment with UV or cholesterol oxide. The ^H/i-iC ratio was compared with the ratio from eontrol cultures har\'ested at the .same time.
RESULTS. Plating efficiencies at hi^h oxygen levels Eollowing the tiiethod of Mitchell, Elgas and Balk (1976) for the selective growth of transformed chick cells, 2 human fibroblast strains and the melanoma lino MM96 were used as a modt-l system to determine whether high oxygen levels would select for tlie growth of transformed human cells compared with normal cells. A difference in plating efficiency was oh.served {Fig. 1), but was too small to be used as a selection method. Attempted transformation The dose-survival respon.sc of several fibroblast strains vi'as determitied to allow cells to be irradiated at known survival levels (Fig. 2). In agreement with the results of Lehmann et a]. (1977), there was no significant difference in sitrvival between the nortnal and melanoma-derived strains. Post-incubation at 40' did not greatly reduce survival, and cholesterol oxide had no cytotoxic effect iu the conceutration range 0-5-10 jLtg/ml (results not shown).
290
P. G. PARSONS AN-D PATRICIA GOSS T
100 -
10 •
_
1
1
20
40
60
80
Percent Oxygen Fig. 1. Effect of tixygen level on tx-Il surviviil expressed as perci'ntage of plating efTic/ifncy in air: O O- MM9e; • Q, BB: A A- MM331-F. Bars ± standard deviation of 6 replicates.
100
10
20
Fluence (ergs x mm" -) Fig. 2. Effect of hyperthermia on UV survival of fibroblasts: O - - - O- MM283-F: • - • - • , MM280-F with 4 h post-incubatioLi at 40°: D D. SGR; • • . SGB with 4 h post-incubation at JO". Bars ±: standard dc\iatioii nf 6 rt'plicates. Not shown when less than twice the .symbol size.
CHOLESTEROL OXIDE AND HUMAN CELLS
291
In one transformation attempt DJM fibroblasts were treated with UV (15 ergs X mm"-), ehole.sterol oxide (10 fig/m\ for 24 h) heat {40' for 4 h) or combinations of these agents. After 1 week the treatments were repeated and the cultures then maintained for 38 weeks. During the second half of this period both control and treated cells heeatne senescent without any evidence of transformation as judged by morphology or multi-layered foci different from controls. The other fibroblast strains were given 5 daily treatments and observed for periods of 4-6 weeks, again with negative results. Chromosome analysis Cultures from transformation experiments in which a UV Huence of 2 ergs X mm~- was used resumed uoruial growth rate after 1 week and were therefore considered suitable for chromosome analysis. Approx. 4-20^ of cells were in metaphase. Most of the abnormalities ( > 95%) were cliromatid breaks or deletions, with a similar average in al! experiments of approx. 1-3 aberrations per scored metaphase. The aberration frequencies resulting from single treatments of 1 normal (1 experiment) and 1 inelanoma-derived fibroblast strain (2 experiments) are sliown in Table 2. All probabilities were calculated on the totals for both strains using the chi-s(iuared test with continuity correction; similar probabilities were obtained by eomparitig means of the weekly values using the t-test. Comparison of results obtained at a single time point usually gave a significant difference, e.^., 1 week after treatment with cholesterol oxide, p < 002 compared with the eontrol. Based on the signifieance levels in Table 2, aberrations were iudueed by cholesterol oxide alone and enhanced by hypcrthermia when cholesterol oxide and UV were used separately and together. Cliolesterol oxide and UV-indueed aberrations were still present after 8 weeks (p < 0()5). Target cultures subjected also to hypertlicrmia initially showed a high aberration frequency which decreased over the 8 week period (p < ()-05). The aberration level iu untreated cells, the number of metaphases counted and the lifespan of the eell strain limit the period over which observations can be made. Thus, Marshall and Scott (1976) found that aberrations induced in normal human fibroblasts by UV (5 ergs X mm--) returned to control levels (7'/) after only 3 days. Most types of treatments gave approx. twice the aberration frequency in the melanomaderived strain as in the normal strain. The relative significance of donor age or melanoma origin conld not be assessed because of the limited number of strains studied. Aberratiou frequencies were also increased by multiple UV heatment of SCB (Table 3), MM185-F and MM288>F fibroblasts (re.sults not shown), but were not significantly increased compared with a single treatment (p > 0-1). DNA repair synthesis For determination of DNA repair synthesis, as judged by sparsely-labelled nuclei iu antoradiographs, separate fibroblast ciiltiucs were treated at higher levels of eholesterol oxide and UV than used iu tlie transformation experiments
292
P. C. PARSONS AND PATRICIA COSS
o c OO
DDO^iOO
o o VV
V V V V VV
( S "*•
293
CHOLESTEROL OXIDE AND HUMAN CELLS TABLE 3. Chromosome aherrcitions in SGB ftbrohlasts jollowiit^ 4 daily treatments. Weeks after treatment
Treatmenf^' Control
1
2
3
3t
-1
2
Total
%
10
2-5
9
17
9-3§
4
21
5-3
8
W
10
7
40
6
7
UV + 40
14
7
10
8
39
9 8§
UV ^ 40 + c o t
20
M
13
6
?6
10 0§
11
treatment levels as Table 2. fNumbers of metaphases with aberrations out of lOO metaphases examined. tOnly one treatment with cholesterol oxide. SSignificance compared with control, p < 0 001.
and liaivt'sted 4 h later. U\' increased the proportion of sparsely-lubelled nuclei (Tahle 4), showing that the method was effective in detecting repair .synthesis. Cholesterol oxide., bnt nt)t cholesterol, was also fonnd to indnee repair synthesis. The efFeet was almost additive with VV in tlie SCB and MM288-F strains (Jitter results not shown) but not in the MM331-F strain. Post-incnbation at 40~ had little effect. TABLE 4. Iinliiclioii of DNA repair synthesis in human fihnihlasts hy cholesterol oxide and UV.
Treatment"
Per cent sparsely-labelled nucleif MM331-F SGB
Control
1-4 It 0-3
1-6 ± 0-28
40
1-9 ± 1-5
2-3 ±
1-8
CO
8-5 ± 0-42
10-6 ±
2-3
CO + 40
10-'* + l)-7
10 0 ± 0-6
UV
8-1 ± 0-4
10-5 ±
3-2
UV + 40
8-9 ± 0-7
11-5 ±
4-4
CO + UV
15-4 + I'4
CO 4 UV + 40
14-6 ±
11
•^Control. 4 b intubation at 3ft" with H) iig/ml cholesterol; 40. 4 h incubation at 40° CO, 4 h incubation with 10 fig/ml cholesterol oxide: UV. 10 ergs x m m - - . fAverage of duplicate slides ± standard deviation (500 cells counted per slide). Significance compared witb control, p < 0 0 1 (except 40° treatment).
294
P. G. PARSONS AND PATRICIA GOSS
Detection of single-strand breaks The potential of alkaline sucrose gradients to resolve single-stranded DNA was improved using a mixture of -^H and "G-Iabelled test and control cells in the same gradient as recotiiniendcd by Dingmaii and Kakunaga (1976). The DNA profiles of strains SGB and M M ' 3 3 1 - F treated with cholesterol oxide for 24 h showed no difference from controls; no cotnbined treatments were used. Tlie same result was obtained when tlie cells were conversely labelled. Using the more sensitive nucleoid sedimentation method, material from UV-irradiated SGB cells (60 ergs x m m - - ) showed greatly rednced sedimentation compared with untreated cells (Fig. 3 ) : in other experiments a dijfference was distinguishable at 10 ergs x turn--, in agreement with Cook and Brazell (1976). Gholestcrol oxide, however, showed no .significant effect in repeated experiments using the SGB and BB strains (Fig. 3), This was also the case when mixtures of labelled cells were analysed in the same gradient.
O
D
0.6
04
0.2
Distance Sedimented Fig. 3. Detection of DNA singlc-strantled breaks in SGB fibrnhlasts by sedimentation of nucleoids (sample zone not sllov^^l). A, imtreittecl cells. B, L h after UV irradiation (60 ergs X m m - - ) . C. 24 h after addition ot cliolesteriil oxide (10 jig/ml), D, O O. untreated cells; • • , 24 ll after addition of cholesterol oxide (10 [ig/ml).
Detection of DNA daimige by inhibition of thymidine
uptake
The melanoma line MM253 was used for tliis test because of its sensitivity to UV (Lavin, Good and C^oitnsilman, 1977) and alkylating agctits (Goss and Parsons, 1977). atid its enhanced ability to incorporate labelled thymidine. A UV dose of 50 ergs x nini-= gave a 90^: decrease in DNA synthesis over 3 h, similar to the response ot HeLa cells reported by Painter (1977); 10 ergs x
CHOLESTEROL OXIDE AND HUMAN CELLS
295
produced a 30% decrease. A 1 h pre-treatment with cholesterol or cholesterol oxide (10 /ig/ml), however, had no significant e£Fect on thymidine ineorporation during this period. DISCUSSION. The present approach to UV transformation was planned as an attempt to circumvent the UV protection systems of human fibroblasts by various means: multiple doses, combined doses with a carcinogenic photoproduct, hyperthermia as a potential promotor, and use of target cells from aged melanoma patients. The lack of sneeess .so far may be due to a requirement for more specific inhibition of repair, or for synergistic factors as yet unknown. The choice of UV wavelength or target cell type should not be limiting factors, since sarcomas are readily induced in mice at this wavelength, the lack of sun-related sarcomas in man being explicable in terms oH thieker skin protection (Blum, 1959). However, closer simulation of the in vivo situation would be desirable, even if less convenient experimentally. Future attempts to transform hnman cells by UV conld be guided by recent experience with rodent cells where use of special contact-inhibited strains, use of phorbol ester (Mondal and Heidelberger, 1976), pretreatment with X-rays (DiPaolo and Donovan. 19761 and critical timing of treatments (Chan and Little, 1976; Mondal and Heidelbergcr, 1976) have led to success in vitro. Appropriate selection methods such as the agar cloning procedure nscd for viral transformation assays may be needed to detect what is evidently a very low transformation frequency. Several new observations, relevant to UV transformation of human cells, were made using carcinogen assays. The induction of chromosome abnormalities and repair synthesi.s by cholesterol oxide provided the first evidence for this compound having a careinogen-Iike effect on human cells. However, more detailed studies of the action of cholesterol oxide may be difficult because of the lack of detectable DNA breaks or other DNA damage. Although the process of UV carcinogenesis may presently be explained in general terms without recourse to an intermediary carcinogen, these results show that cholesterol oxide and other skin photoproducts should be further evaluated as potential carcinogens m man. The synergistic eflFect of hyperthermia on chromosome aberrations supports previous evidence (Goss and Parsons, 1976; Freeman and Knox, 1965) that increased skin temperature may play a role in UV carcinogenesis. The observation that some damage from both cholesterol oxide and UV remained at 8 weeks suggests that factors synergistic to the later stages of carcinogenesis could be effective long after UV exposure. Epidemiological studies of such factors would need to consider this possibility. Acknowledgements. The co-operation of Dr. J. H. Littk- and Dr. N. C. Davis, of the Quffiisland Melanoma Project, Princess Alexandra Hospital. Brisbane, is gratefully acknowledged. We wi.sh to thank Leanne Morrison and Juanita Yuen for excellent technical assistance. This work was supported by the Queensland State Government, and assisted by a grant from the National Health and Medical Research Council, Canberra.
296
P. C. PARSONS AND PATRICIA GOSS
REFERENCES. ABHONDANDOLO, A. (1977): 'Pro.spccts for CKJSS, P.. and PARSONS. P. G. (1977): evaluatinjf genetic damage in mammalian 'The efFect of hyperthermia and melcells in culture.' Mtitat. Res.. 42. 279. pbalan ou survival of human fibroblast strains and melanoma cell lines.' Cancer BHATTACHARYYA, A. K., CONNOR, W . E.. Res., 37. 152. and SPECTOR, A, A. (1972): 'Excretion LAVIN, M. F . , GOOD. M., and CO"NSILof sterols from ths skin of normal and MAN. C. (1977): 'Resistance of human hypercholcsterolemic humans.' J. Clin. melanoma cells to ultraviolet and ionizInvest., 51, 2060. ing radiation.' Proc. Au.st. Biochem. Soc, BLACK. H . S., and Lo. W. (1971): Torma10, 77. tion of a carcinogen in human skin LEHMANN, A. R.. KIKK-BELL. S., ABLETT, irradiated with ultraviolet light' Nature. C. F., HARCOURT. S. A., DE WEERD234, .306. K.'vsTEi.EiN. E. A.. KEIJZER, W . . and BLUM, 11. F. (1959): "Carcinogene.sis by HALL-SMITH. P, (1977 ): 'Repair of ultraUltraviolet Light", Princeton University \iolet liglil damage in a variety of human Press. Princeton. fibroblast cell strains.' Cancer Res., 37, 904. CHAN. G . L.. and LITTLE, J. B. (1976): MARSHALL. R. R.. and SCOTT. D . (1976): 'Induction of oncogenic transformation 'The relationship between chromosome in vitro by ultraviolet light." Nature. damage and cell killing in UV-irradiated 264, 442. normal and xeroderma pigmentosum cells.' CouKN, M. M.. and IIIHSCHOHN. K. (1971): Mtitat, Res.. 36, ,397. 'Cytogenetic studies iu animals.' In MITCHELL, R, S.. FLGAS, R. J., and BALK, "Chemical Mntagens. Principles and S. D. (1976); 'ProliferaHon of Rous Method.s for Their Detection", A. Holsarcoma virus-infected, but not of Iaender (editor). Plenum Press. New nonnal. chicken fibroblasts in oxygenYork, \'ol. 2, p. 515. em iched environment; preliminary reCOOK, P. R., and BRAZELL, I, A. (1976): port.' Proc. Nat. Acad. Sci. (U.S.A.). 73, 'Detection and repair of single-strand 1265. breaks in nuclear DNA.' Nature. 263. MoxDAL, S.. and HEIDELBERGER. C, (1976): 679. 'Transformation of C3H/10T1/2CL8 DiNGMAN. C. W., and KAKUNACA. T . nionse embryo fibroblasts by ultraviolet (1976): 'DNA strand breaking and reirradiation and a phorbol ester.' Nature. joining in response to ultraviolet light 260. 710. in nonnal human and xeroderma pigMooRHKAD. P. S., and NOWELL, P. C, nientosum cells.' Int. ]. Radicit. Biol. 30. (1964); 'Chromosome cytology." In 55. "Methods in Medical Research'", H. N. DIPAOLO, J. A., and DONOVAN, P. J. Eisen (editor). Year Book Medical Pub( 1976): 'In vitro morphologic translishers, Chicago, Vol. 10, p. 310. formation of Syrian hamster eells by PAINTER, R. B. (1977): 'Rapid test to l^V-irradiation is enhanced by Xdetect agents that damage human DNA." irradiation and imadected by chemical Nutiire. 205. 650. carcinogens.* Int. ]. Radiat. Biol, 30, 41. STICH. H . F . , LAM, P., Lo. L. W., KOROFiESKR, L, F., and FU:SEH. M . (1967): PATNTCK. D. J., and SAN, R. II. C. "Reagents for Organic Synthesis". Vol. 1. (1975): 'The search for relevant shortJ. Wiley and Son.s. New Y'ork, p. 136. term hioassays for chemical carcinogens: FREEMAN, R. G., and KNOX. J. M. (1965): th? tribulations of a modern Sis\'phus.' The factor of temperature in ultraviolet Can, } . Gcnt't, Cijiol. 17. 471. injury.' Arch. Environ. Health. 11, 477. UitHACH, F. (1975): 'Ultraviolet radiation: Goss, P., and PARSONS, P. G. (1976); interaction with biological molecules.' In 'Temperature-sensitive DNA repair of "Cancer, a comprehensive treatise"'. F. ultraviolet damage in hujuan cell lines.' F. Becker (editor). Plenum Press, New Inf. /. Cancer, 17. 296. York, Vol. 1. p. 441.
