Enhanced Photoproduct Repair: Its Role in the - Cancer Research

[CANCER RESEARCH 55, 181-189, January 1. 1995]

Enhanced Photoproduct Repair: Its Role in the DNA Damage-Resistance Phenotypeof Human Malignant Melanoma Cells1

David H. Hatton,2 David L. Mitchell, Paul T. Strickland, and Robert T. Johnson

Cancer Research Campaign, Mammalian Cell DNA Repair Research Group, Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, Uniteci Kingdom fD. H. H.,R. T. J.Â¡;Department of Carcinogenesis, M. D. Anderson Cancer Center, Science Park, Research Division, Smilhville, Texas 78957 [D. L M.J; and Department of EnvironmentalHealth Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205 Â¡P.T. S.]

ABSTRACT

A fundamental issue in understanding melanoma is to seek the basis forthe cellular resistance to DNA damaging agents, which is manifested invivo as pronounced tumor resistance to therapeutic agents. The publishedconsensus on melanoma has been that exaggerated postreplication recovery (PRR), rather than excision repair, underlies the unusual damage-

resistance phenotype. We examined the resistance to the model DNAdamaging agent, UV-C, of subclones derived from a human metastatic

melanoma cell line. The clones essentially fall into two groups: one withnormal and the other with enhanced resistance. We exploited this range toinvestigate the interrelationships between replication, transcription, andrepair of DNA after UV irradiation. Subclones resistant to UV killing wereindeed found to possess enhanced rates of PRR and were coresistant tocisplatin. However, we now report an overall elevation of photoproductrepair in both melanoma groups compared to nonmelanoma controls andconclude that this accounts for the resistant melanoma phenotype, including that of enhanced PRR. Repair enhancement may explain chemoresis-

tance, while loss of efficiency of certain functions, such as PRR, due to theintrinsic genetic lability of tumor cells, may generate the class of melanoma subclones exhibiting only normal resistance.

INTRODUCTION

Some human tumors are characterized by their considerable resistance to killing by DNA damaging agents. Moreover, the degree ofresistance to a particular agent may vary from one tumor sample toanother and among different clones of the same tumor (1, 2). Suchheterogeneity offers useful material to investigate correlations between resistance and the important determinants of survival, e.g.,DNA repair and PRR3 functions, and, ultimately, to address whether

the acquisition of resistance is a consequence of one or more of thegenetic changes leading to the transformed phenotype.

Human malignant melanoma is a serious neoplastic disease of thepigmentary system which afflicts a broad spectrum of age groups, andamong westerners is one of the most rapidly increasing forms ofmalignancy (3). This rising prevalence is particularly worrying giventhat it metastasizes early in its history and has a poor prognosis. Thereis currently no effective therapy for metastatic melanoma due to itsrefractory nature toward chemotherapy, radiotherapy, and immuno-

therapy (4).The poor response rate of metastatic melanomas to chemotherapy

by nitrosoureas, VÃ¬ncaalkaloids, cisplatin, dacarbazine, and melpha-

lan (5) is suggestive of a broad underlying resistance to the effects ofDNA damage. Melanoma tumors have long been recognized as displaying resistance to radiotherapy (6) and this is mirrored by their in

Received 8/29/94; accepted 10/31/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Cancer Research Campaign, of which R. T. J. is a Research

Fellow.2 To whom requests for reprints should be addressed.^ The abbreviations used are: PRR, postreplication recovery; (6-4), pyrimidinc-

pyrimidone (6-4) photoproduct; ara-C, 1-ÃŸ-D-arabinofuranosylcytosine; ASG, alkalinesucrose gradient; BNDC, benzoylated naphthoylated diethylaminoethyl cellulose; CPD.cyclobutane pyrimidine dimer; HAP. hydroxyapatite; HU. hydroxyurea; NER. nucleotideexcision repair; NET, 1 M NaCI, 0.1 mM EDTA, 10 mM Tris-HCI. pH 7.5.

vitro resistance to ionizing radiation (7), although there is a wide

spectrum of response encompassing both the normal and the veryresistant (8). The repopulation of tumors after radiotherapy or chemotherapy has been attributed to the survival of a resistant variantsubpopulation within the primary tumor. This situation may be particularly common among human melanomas which demonstrate highlevels of repair of potentially lethal damage (1).

Taken as a whole, there are surprisingly few accounts of thesensitivity of human melanoma cells in vitro to DNA damagingagents, although a number of observations (9-11) point to a highdegree of UV-C resistance among these cells. A favored damagingagent for numerous in vitro studies of human cells has been 254-nmUV-C radiation, which maximally induces dimerization of adjacentpyrimidines in DNA. forming CPD and pyrimidine-pyrimidone (6-4)photoproducts (12). Such lesions, in common with those helix-dis

torting adducts formed by reaction of chemotherapy agents, such ascisplatin, with DNA, are repairable by the major mammalian DNArepair pathway of NER.

In the face of limited photoproduct removal data which did notindicate enhanced repair in melanomas (10, 11), it was suggested thatboth murine and human melanoma cells possess an enhanced abilityto replicate their DNA on a UV-damaged template (9, 11, 13), giving

them a survival advantage through increased rates of PRR, which isthe process by which maturation of newly synthesized DNA, into highmolecular weight forms devoid of discontinuities, is achieved on adamaged template (for review, see Ref. 14).

In this article, we demonstrate the variation in susceptibility tokilling in vitro by the archetypal DNA damaging agent, UV-C, of

clones derived from the human metastatic melanoma line RVH421.We find that the clones essentially fall into two groups: one withsurvival values typical of other tumor cells (EJ30, HeLa) and onewith much greater resistance. We also confirmed that this range of

sensitivities is typical of other genetically independent melanomas.In accord with previous reports, we find that UV-resistant linesdisplay a damage-resistant phenotype including enhanced rates of

PRR following UV irradiation. However, we now report thatUV-induced DNA incisions (a measure of NER activity) are uni

formly high in all our melanoma lines, regardless of their sensitivity, compared to nonmelanoma human tumor controls, and thisis mirrored by their exceptional rates and extents of both (6-4)

photoproduct and CPD repair. These repair measurements suggestthat we should reinterpret the observation of enhanced PRR as aby-product of the exaggerated DNA repair processes in melanoma

cells, leaving a less damaged template to interrupt replication. Wealso present evidence that the sensitive class of melanomas expressa recessive defect, seen as reduced PRR, and their cellular resistance to UV is restored in hybrids derived by crossing sensitive withresistant clones.

Taken together with an additional observation of the existence ofcoresistance to both UV and cisplatin, our results are important inproviding a plausible explanation for the origins of melanomachemotherapy resistance via enhanced repair.

181

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ENHANCED HUMAN MELANOMA PHOTOPRODUCT REPAIR

MATERIALS AND METHODSl'eli Lines and Culture Conditions

The following cell lines were used: (a) RVH421, a human melanoma cellline derived from a cranial metastasis (15), kindly provided by Professor HenryHarris, Sir William Dunn School of Pathology (Oxford, United Kingdom);(b) FM, a human melanoma line (CRL1424, obtained from ICN Flow) received at passage 28; (c) A375, a human melanoma cell line (CRL1619,obtained from the American Type Culture Collection); (d) EJ30, a diploid cellline derived from a human bladder carcinoma (generous gift from Dr. R. J.Hastings, Department of Cellular Pathology, Imperial Cancer Research Fund,London, United Kingdom); and (e) HeLa, derived from the original Denver(Denever, CO) stock of Dr. T. T. Puck.

Subclones R1-R24 were derived from the RVH421 cell line. RVH421 cellswere plated out at low density into 90-mm dishes and independent colonies

were identified visually 2 weeks later. Randomly selected subclones wereestablished by harvesting cells from these colonies.

All cell lines were grown in Eagle's MEM buffered with bicarbonate and

supplemented with 4 mM glutamine, 50 units/ml streptomycin, nonessentialamino acids, and 10% PCS (ICN Flow). Cultures were incubated at 37Â°Cin

3% C02 in humidified incubators and were split 1:3 every 3â€”4days, the cells

being detached with pancreatin (GIBCO Europe Ltd). Stocks were maintainedfor 3 to 4 months before returning to freshly thawed cultures. The followingradiochemicals were used: [merfry/-3H]thymidine (55 mCi/mmol), [methyl-14C]thymidine (57 mCi/mmol), and [Â»Â¡Â£r/iv7-3H]uridine(28 mCi/mmol), all

purchased from the Radiochemical Center, Amersham.

Production and Selection of Melanoma Hybrids

Melanoma clones bearing either the ouabain-resistance marker (selected forby growth in 5 X 10~7 M ouabain; Sigma) or the neomycin-resistance marker

(electroporated into the cells and selected for by growth in medium containing600 (Ag/rnl G418) were fused by Sendai virus (16), and hybrids were selectedin medium containing both selective agents.

UV-Irradiation and Clonal Cell Survivals

Irradiation was carried out using a Philip's germicidal tube emitting light

largely at 254 nm. The tube was calibrated with a Radiometer (model ILI 700International Light Inc., Newburyport, MA). Dose rates were selected todeliver desired doses in periods of 5-30 s.

Clonal UV Survival. Cells were harvested from log-phase cultures andirradiated while suspended in PBS-1% PCS at a density of 105/ml, then plated

at predetermined densities in duplicate 60-mm culture dishes (Falcon Plastics)in complete medium. After 2-week incubation the cells were fixed, stained

with Giemsa, and colonies containing 50 or more cells counted.Clonal Cisplatin Survival. Appropriate numbers of cells were seeded in

triplicate into 60-mm dishes and the following day were exposed to serum-freemedium containing c/.s-diamminedichloroplatinum(II) (Sigma) for l h at 37Â°C.

The medium containing the drug was then removed, and the cells were washedtwice with PBS, then incubated in complete medium for another 2 weeks forcolony formation.

Assay of Cyclobutane Pyrimidine I timers by Ion Exchange

Chromatography

Cells were prelabeled in 90-mm dishes for 48 h in medium containing 0.1/xCi/ml [mer/Â¡y/-3H]thymidine, then incubated another 24 h in unlabeled me

dium. Cells were harvested, counted, and UV irradiated in PBS, then spundown and frozen as a pellet at â€”20Â°C.Dimers were assayed according to the

method of Sekiguchi et al. (17).

DNA and RNA Synthesis Recovery Assays

At appropriate times after UV irradiation or sham irradiation, cells induplicate dishes were pulse labeled in medium containing 1 /iCi/ml [methyl-3H]thymidine (DNA synthesis assay) or 2 ju,Ci/ml [mef/iv/-3H]uridine in 10%

dialyzed serum (RNA synthesis assay) for 30 min, thus permitting equilibration with the soluble pools. Measurement of the soluble pools and the acid-

precipitable incorporation was performed as described previously (18). Rela

tive incorporation was derived by calculating trichloroacetic acid/NaOH countratios, expressed as a percentage of the values obtained for unirradiatedcontrols.

BNDC Postreplication Recovery Assay

Cells were irradiated in duplicate and incubated for l h to allow terminationof replicons active at the time of irradiation, then pulse labeled with 10 fÂ¿Ci/ml[mei/!>7-3H]thymidine for 10 min. After washing with warm PBS the cells wereincubated for the appropriate chase period in medium containing 10~5 M

deoxyribonucleosides, then lysed and prepared for Chromatography as described by Pillidge et al. (19). BNDC Chromatography columns were preparedaccording to the method of Strauss (20) and run as described by Pillidge et al.(19). Briefly, after column washing, double-stranded DNA was eluted intovials with three washes of 1.5 ml l M NET and DNA with single-strandedregions eluted by three washes with 2% caffeine in l M NET at 45Â°C.To

display the results, the l M NET counts were expressed as a percentage of thetotal counts (1 M NET plus 2% caffeine in l M NET). This gave an estimate ofthe percentage of labeled DNA fragments that appeared to be double-stranded.

Alkaline Sucrose Gradient Postreplication Recovery Assay

Cells were irradiated in situ and pulse labeled with 10 /j,Ci/ml [methyl-3H]thymidine for 10 to 60 min, depending on the UV dose, the label was

removed, and the cells were incubated for the appropriate chase period. At theend of the chase period the dishes were then washed twice in ice-cold PBS and

the cells were scraped into suspension in 0.5 ml PBS. The cell suspension(0.1 ml) was lysed on the surface of a 4.8-ml linear alkaline sucrose gradient(5-20% sucrose, 0.9 M NaCl, 0.01 M EDTA, 0.1 M NaOH) in a surface lysis

layer of 0.5 M NaOH, 0.02 M EDTA, and 0.1% NP40 in Beckman Ultra-Cleartubes. Lysis occurred at 22Â°Cin the light for 1 h, after which the gradients werespun at 38,000 rpm at 20Â°Cin a SW50.1 rotor in a Beckman L7-65 ultracen

trifuge. Gradients were fractionated onto 30 GF/C filters, washed twice with5% trichloroacetic acid and once with 95% ethanol, dried, and counted in 3 mlscintillation fluid (Optiscint HiSafe).

Assay for Incisions by HAP chromatography

HU (10~2 M) and ara-C (10~4 M) were added to each dish of uniformly

[mer/!v/-3H]thymidine-labeled cells for 40 min prior to irradiation. Medium

was removed and the cells were UV irradiated in PBS containing the sameconcentration of HU and ara-C. The cells were returned to prewarmed mediumcontaining HU and ara-C and incubated for various periods, then lysed ac

cording to the method of AhnstrÃ¶m and Erixon (21), as described by Squireset al. (22), except that sonication was used to reduce the size of the DNA (8 s,24 KHz, 8-iun amplitude; MSE Instruments Ltd). The DNA unwinds in alkali

to a degree that is a function of the frequency of DNA breaks held open byara-C and HU. The proportion of single-stranded DNA, as a result of unwind

ing in alkaline sucrose, was determined by HAP chromatography. The numberof DNA breaks was estimated from a calibration curve derived from cells thathad been X-irradiated to introduce known numbers of DNA breaks (2.5/109

daltons/krad; Ref. 23) before processing and HAP chromatography.

(6-4) Photoproduct and Cyclobutane Dimer Immunoassays

Cells were uniformly prelabeled by incubation in medium containing 0.1/xCi/ml [wef/ry/-14C]thymidine for 3 days, returned to normal medium, then

PBS washed and UV irradiated in situ the following day. The Strickland assayused unlabeled cells. After the appropriate repair interval, cells were harvestedby scraping in a small volume of lysis solution (10 mM Tris-HCl, 1 mM EDTA,150 mM NaCl, 0.4% SDS), then incubated overnight at 37Â°Cin lysis solution

containing 0.3 mg/ml proteinase K (Boehringer Mannheim). DNA was extracted and ELISAs performed as described by Strickland et al. (24) or RIAsperformed as described by Mitchell and Clarkson (25). The Strickland ELISAsused a polyclonal rabbit antiserum that recognizes primarily T(6-4)C and alsothe rare T(6-4)T photoproducts4 and the Mitchell RIAs involved use of a

polyclonal rabbit antiserum recognizing T(6-4)C photoproducts and ToT

cyclobutane pyrimidine dimers (25, 26).

4 Unpublished data.

182




RESULTS

Derivation of Subclones and Determination of UV Sensitivity

In our early passages of the metastatic melanoma line RVH421there was considerable heteroploidy; from this culture we derived anumber of randomly selected subclones (R1-R24). The parentalRVH421 population eventually stabilized as a stable near-triploid cell

line with a mean of 64 chromosomes. No direct selection for UVresistance or sensitivity was applied during the derivation of RVH421or any of the 24 subclones derived therefrom.

Each subclone (R1-R24) was screened for its resistance to UV

irradiation in comparison to both the parental RVH421 line and acontrol human bladder carcinoma cell line (EJ30) which has normalUV resistance (22). Colony UV survivals (Fig. L4) showed that theclones generated from RVH421 displayed a spread of UV resistanceclustering at both near-normal (e.g., R13, R16, and R24) and su

pranormal levels (e.g., RIO and R7). That this range of sensitivities istypical of other, genetically independent, melanomas was confirmedin the FM and A375 cell lines (high and normal UV-C resistance,

respectively, Fig. IA).Two melanoma subclones, R13 and RIO, representing the two

extremes of sensitivity, along with the parental RVH421 line and thecontrol tumor line EJ30, were chosen for subsequent detailed analysis.The results from R13 and RIO are considered to be representative ofthe two survival classes. The survival parameters Dq, D(), and Du)

100

Table 1 Survival parameters of selected tumor cell lines

10 15 20 25UV dose (J/sq.m)

30 35

Fig. 1. A, normal and elevated UV resistance revealed by colony survival. RVH421(â€¢),RIO (D), R13 (O), R24 (x), R16 ( + ), R7 (â€¢),EJ30 (A), FM (A), and A375 (Â«) cellswere harvested from proliferating cultures, UV irradiated in PBS-1% FCS, and appropriate numbers plated out. After 2-week incubation, colonies were fixed, stained, andcounted. Shows the survival of irradiated cells expressed as a percentage of the survival(i.e., plating efficiency) of unirradiated controls. Points represent the means of four to sixindependent determinations. The line of best fit was calculated by least squares analysis.ÃŸ,UV resistance of R13 restored in hybrids. Proliferating hybrids R13 X R13 [O, n (no.of independent hybrids) = 2], R13 X RVH421 (A, n = 11), R13 X RIO (D, n = 3),RVH421 X RVH421 (â€¢.n = 3), and RVH421 x RIO (A, n = 3) were assayed for UVresistance by colony survival as described in A. The ouabain- and G418-resistant mela

noma clones used in production of hybrids had the same degree of UV resistance as theparents from which they had been derived (data not shown).

SurvivalparameterD

"â€¢*DO"

D,,,"RVH4214.0

Â±0.813.5Â±0.3

25.8RIO7.1

Â±1.014.5Â±0.4

24.1R130.0

Â±1.32.7Â±0.4

8.0EJ303.1

Â±0.57.1+0.2

12.3" Survival parameters: all values are in units of J/m2. Each was calculated from the

lines of best fit. Standard errors are given for the Dq and D0.h Dq, extrapolated dose at which survival remains at 100%, a measure of the shoulder

of the survival curve; D((, mean lethal dose, giving on average one lethal hit per cell; D1(),dose required to reduce survival to 10%.

100

01 23456789 10Cis-Pt dose (microgram/ml)

Fig. 2. UV-resistant melanoma cells express coresistance to cisplatin. RVH421 (â€¢),RIO (D), and R13 (O) cells were seeded into dishes and allowed to attach overnight.Medium was then removed and the cells were exposed to serum-free medium containingcisplatin (ci'j-Pt) for 1 h. The drug was removed, and the cells were washed twice in PBS.

then incubated in complete medium for 2 weeks. Colony formation by drug-treated cells

was compared to untreated controls. Points, means of three samples.

(calculated from the survival curves), which are measures of susceptibility to UV killing, of these four cell lines are given in Table 1. Itis apparent that RVH421 and RIO are both UV-resistant lines, each

having a larger Dq and D()/D10 values twice that of FJ30, while R13has a slight UV sensitivity compared to the nonmelanoma control.

It is also clear that the pattern of UV resistance among the melanoma clones was mirrored by their cellular sensitivity to a shortexposure to the chemotherapy agent cf's-diamminedichloroplatinu-

m(II). Fig. 2 demonstrates that the cisplatin resistance of the UV-

resistant lines RVH421 and RIO is pronounced compared to that ofthe UV-sensitive subclone R13.

There is also an apparent relationship between the UV resistance ofthe RVH421 melanoma clones and their ploidy: all lines with increased UV resistance were triploid (e.g., RVH421) or tetraploid (e.g.,RIO), while lines with reduced resistance were near-diploid (e.g., R13and R16) (data not shown). Polyploidy is a well-known characteristic

of tumor cells and malignant melanoma is no exception (27), but it isknown that polyploidy per se does not lead to increased UV resistancein other cell types through a gene dosage mechanism (10), andpolyploid HeLa and EJ30 cells do not show enhanced UV resistance.5

As a test of the dominance/recessiveness of the melanoma pheno-

type a series of permanent hybrid lines was constructed by cell fusion.Self-crossing R13 (R13.neoR X R13.ouafeR) produced no change in

UV sensitivity. However, whenever R13 was fused with a moreresistant melanoma partner (RVH421 or RIO) the resulting hybrid wasalmost as resistant as the original resistant partner (Fig. IÃŸ).Thus thephenotype of R13 is recessive and suggests that it has lost a functionimportant to survival.

5 D. H. Hatton. D. L. Mitchell. P. T. Strickland, and R. T. Johnson, unpublished

observations.

183




Role of Photoprotection: Degree of Pigmentationand Yield of Cyclobutane Dimers

It is possible that while RVH421 is essentially amelanotic, subtledifferences in melanin content among the clones might affect the yieldof photoproducts. Measurement of CPD yield in RVH421-derived

clones showed little variation (data not shown). Furthermore, dimerinduction in these amelanotic melanoma lines was equivalent to thatin EJ30 and hence does not explain the observed differences in UVsurvival.

Rates of Nucleic Acid Synthesis Post-UV Irradiation

A characteristic response of mammalian cells when treated withUV and other DNA damaging agents is a rapid, although temporary,dose-dependent inhibition of the rate of DNA synthesis, due to the

inhibitory effect of DNA lesions on rates of initiation (28) andelongation (29).

DNA Synthesis Recovery. We investigated the inhibition andrecovery of DNA synthesis by pulse-labeling cells with [3H]thymi-

dine at intervals after irradiation. Each line displayed dose-dependent

inhibition, yet while RVH421 and RIO recovered normal rates ofsynthesis 11 h after receiving 5 J/m2 (Fig. 3, A and B), the DNA

synthesis of R13 and EJ30 remained severely inhibited after this doseand only partially recovered following 2.5 J/m2 (Fig. 3, C and D).

These results show that the cell lines most resistant to killing by UVirradiation, RVH421 and RIO, possess relatively UV-resistant DNA

synthesis and enhanced rates of recovery.RNA Synthesis Recovery. In addition to replication, transcription

is temporarily blocked by pyrimidine dimers in template DNA (30).The depression and recovery of RNA synthesis following UV irradiation was investigated. In each cell line RNA synthesis was depressedrapidly, to the same degree, after equivalent low doses of UV irradiation; however, RVH421 and RIO recovered normal rates of synthesiswithin 7 h of doses of up to 5 J/m2, while R13 and EJ30 only showedcomplete recovery in that time following 2.5 J/m2 (data not shown).

We conclude that, as for DNA synthesis recovery, the UV-resistant

lines possess superior rates of recovery of RNA synthesis.A link has previously been established between UV survival and

rates of macromolecule synthesis in investigations of excision repair-deficient (31) and PRR-defective cell lines (32). Given these associ

ations, our results can only be more fully interpreted after examiningrates of both PRR and photoproduct repair.

PRR

The discovery of UV-sensitive mammalian cell lines that have

apparently normal excision repair but are defective in their abilityto synthesise DNA on a damaged template (19, 33, 34) suggests animportant relationship exists between PRR and survival after UVtreatment. We looked for enhanced PRR in our UV-resistant

lines by means of (a) DNA chromatography on BNDC and (b)ASGs.

The rates of maturation, in the absence of DNA damage, monitoredby BNDC chromatography, were normal in each cell line (Fig. 4,unirradiated controls). However, over a wide range of UV doses andchase times, the UV-resistant lines RVH421 and RIO consistently

showed a stronger PRR phenotype compared to the control EJ30 (Fig.4, irradiated samples). By contrast, the PRR of R13, which hasEJ30-like UV survival, showed no such enhancement (Fig. 4C). By

calculating the initial slopes of the maturation curves, the PRR ratecan be estimated over the first hour of chase after irradiation. Theresults indicated a 2-fold enhancement of PRR in the UV-resistant

lines RVH421 and RIO compared to R13 and the EJ30 control(Table 2).

'S100"5A

zD io 12

2 4 6 8 10Time (hrs post irradiation)

12

Fig. 3. Enhanced recovery of DNA synthesis in UV-resistant melanoma cells. RVH421(A), RIO (ÃŸ), R13 (C), and EJ30 (D) cells were UV irradiated (2.5 J/m2, â€¢;5 J/m2, D;10 J/m2, A) in situ and either pulse labeled immediately in medium containing [3H]thy-

midine for 30 min or incubated for specified periods before being pulse labeled. Cellswere extracted first with trichloroacetic acid, then with NaOH and the radioactivity in eachfraction was determined. The ratio of acid- and alkali-soluble counts was determined ateach point and expressed as a percentage of that ratio determined in unirradiated controls.Points, means of duplicate samples.

It could be argued that the enhanced PRR of RVH421 and RIO isthe result of a special response of the replication machinery subsequent to UV irradiation, this being the initiation of replication inundamaged regions of the genome. This hypothesis was tested in

184




12345Time (his post irradiation)

Fig. 4. Time course of maturation of DNA post-UV assayed by BNDC chromatography: elevated efficiency of UV-resistant cells. Log-phase cells (RVH421, A; RIO, B; R13,C; and EJ30, D) were washed with PBS, irradiated (5 J/m2, D; 10 J/m2, A) or sham

irradiated (â€¢)in situ, then returned to medium. Dishes were incubated for 1 h, then thecells were pulse labeled with [3H]thymidine for 10 min and chased by further incubation

in nonlabeled medium for various times before processing for BNDC chromatography.Shows the counts due to radioactivity eluted in the l M NET double-stranded (d.s. )fractions expressed as a percentage of the total counts eluted in the single- and double-

stranded DNA fractions. Points, means of duplicate samples.

experiments in which the ability to mature a pulse of label deliveredjust prior to irradiation was determined (Fig. 5). RVH421 cells stillmatured the label which had been delivered prior to irradiation at thesame rate as if the label were given l h post-UV, and the same result

was achieved with EJ30 cells given an equitoxic dose. Thus, selectivereplication of undamaged portions of the genome does not account forthe observation of enhanced PRR.

The BNDC results described above were corroborated by use ofASGs which reveal maturation of nascent DNA molecules postir-

radiation. As expected, all unirradiated cells showed no differencesin their abilities to mature low molecular weight species intoparental forms within a 1-h chase period, confirming their equiv

alent rates of replicÃ³n joining (data not shown). However, theUV-resistant lines RVH421 and RIO matured to almost parentalsize a pulse of label given after irradiation with 15 J/m2 (Fig. 6A),

while R13 and EJ30 showed poor maturation, with radioactivity beingsmeared across the gradient, even after a lower dose of UV (10 J/m2, Fig.

6, C and D). These results remain consistent with those produced by theBNDC method, indicating that PRR is enhanced in both RVH421 andRIO compared to R13 and EJ30. We confirmed the general applicabilityof these results by finding a similar enhancement of PRR, using bothBNDC and ASG techniques, in two additional UV-resistant lines (sub-clone R7 and the independent melanoma line FM, Fig. 6ÃŸ)while non-enhanced (R13-like) PRR was found in the UV-sensitive subclone R16

(data not shown).

Table 2 Rates of UV-induced PRR are enhanced in UV-resistant melanoma lines

UV dose(J/m2) RVH421" RIO" R13" EJ30"

:10

3427.5

3025.1

1611

2211.25

" Rates were calculated from the initial slopes (during the first hour of chase) of the

BNDC maturation curves for each cell line. Values are expressed as: A (percentage countsin double-stranded fractions)/!!.

12345Time (hrs post labelling)

Fig. 5. No effect from different labeling protocols on the time course of DNA maturation.RVH421 (A) and FJ30 (B) cells were either pulse labeled immediately before irradiation (15J/m2, A; 5 J/m2, B) and chased for various periods thereafter (D); or pulse labeled and chased,

beginning l h after irradiation (A), then processed for BNDC chromatography. â€¢maturationof unirradiated controls. Points, means of duplicate samples.

185




14-

10 20Fraction no.

Fig. 6. Pulse-chase alkaline sucrose gradient profiles of daughter strand DNA: efficientkinetics of maturation in UV-resistant cells. Log-phase cells (RIO, A; FM, B: R13, C; andFJ30, D) were irradiated in situ (15 J/m2, A and ÃŸ;10 Â¡Im1,C and D), incubated for l h,the cells pulse labeled with [3H]thymidine for 60 min, and processed either immediately

for ASG chromatography (G) or after being chased by further incubation in nonlabeledmedium for 4 h (â€¢).Points, counts due to radioactivity in each of the 30 ASG fractionsexpressed as a percentage of the total counts in the gradient. RVH421 produced a resultindistinguishable from RIO (A) (data not shown).

DNA Repair

UV-induced Incision Activity. UV photoproducts are substratesfor excision repair enzymes, the DNA incising activity of which can

be determined quantitatively by the accumulation of DNA strandbreaks in the presence of inhibitors of DNA repair synthesis, revealedby alkaline unwinding and HAP chromatography (21).

The three melanoma lines RVH421, RIO, and R13 were equallyproficient over a range of UV doses (1-8 J/m2) in incision activity

measured immediately postirradiation. At the higher UV doses, themelanoma lines each produced approximately double the number ofbreaks of the control EJ30 and also of a HeLa control (Fig. 7),indicating that, under these conditions, their repair is less easilysaturated. Thus the melanoma lines have an initial incision capacitythat is quite distinct from the other transformed lines.

When UV-induced DNA break accumulation was reversed, by

removal of the DNA synthesis inhibitors, all of the cell lines rapidlyresealed the breaks, thus, regardless of sensitivity, all were equallyproficient in performing the repair synthesis and ligation events necessary for repair patch completion (data not shown).

Immunological Determination of Repair. An analysis of repairof photoproducts was accomplished by use of anti-(6-4) and anti-

CPD antibodies in a series of immunoassays. Both the Strickland (Fig.SA) and Mitchell (Fig. 8ÃŸ)assays revealed very rapid rates of (6-4)

repair in each member of the RVH family (including R13) while, bycomparison, repair by EJ30 cells was slow and incomplete following20 J/m2 (Fig. 8A) and was slow, although eventually completed at halfthe melanoma rate, following 10 J/m2 (Fig. 8fl). Thus, these data are

in agreement with the HAP incision experiments, showing a 2-fold

enhancement of early repair events in the individual melanoma celllines. Two independent human melanoma cell lines (FM and A375),possessing different degrees of UV resistance, had a repair efficiencysimilar to the RVH family in the Mitchell assay, removing 80% ofbinding sites in l h (Fig. 8B).

Irradiated human cells remove CPDs from the overall genome at aconsiderably slower rate than that reported for (6-4) photoproducts(12, 35-37). In a series of experiments, the Mitchell anti-ToT

antiserum revealed surprisingly rapid and complete repair in all members of the RVH family (70% repair in 12 h; Fig. 9) and broadlysimilar rates in the independent FM and A375 melanoma cell lines, incontrast to that observed in both EJ30 and HeLa controls (50% repairin 24 h; Fig. 9).

14 -[

20 40 60

Time (mins post irradiation)so

Fig. 7. High UV-induced DNA incision capacity of melanoma cells. RVH421 (â€¢),RIO(D), R13 (O), EJ30 (A), and HeLa cells (A) which had been uniformly [3H]thymidine

prelabeled were split into culture dishes and incubated overnight. The cells were thenpreincubated in medium containing the inhibitors HU and ara-C for 40 min before beingUV irradiated in siiit with 4 J/m2, and returned to medium containing the inhibitors for

specified periods before alkaline lysis and HAP chromatography. Points, means ofduplicate samples and corrected for the number of breaks (range, 0.0-1.0) found insham-irradiated samples of each cell line. The differences in break accumulation were not

attributable to differences in efficiency of inhibition of repair patch formation (due todifferences in efficiencies of phosphorylation of ara-C), since identical results wereobtained when aphidicolin was substituted for ara-C (data not shown).

186




100

0.0 0.5 1.0 1.5 2.0 2.5Time (hrs post irradiation)

Fig. 8. Rapid removal of antibody binding sites associated with (6-4) photoproducts

in melanoma cell lines. RVH421 (â€¢),RIO (D), R13 (O), EDO (a), FM (A), and A375 (+)cells were irradiated in situ with 20 J/m2 (A) or 10 J/m2 (ÃŸ)and either processed

immediately or after incubation in normal medium for up to 24 h (Strickland assay. A) or3 h (Mitchell assay, B). DNA was extracted from each sample and antibody binding siteswere assayed by ELISA (A) or RIA (B). Points, means of duplicate determinations ofsingle samples (A) or means of two to three assays of independent samples performed induplicate (ÃŸ);bars, SEM. Each point is expressed as a percentage of the inhibition presentin samples not permitted to repair.

DISCUSSION

We have exploited the clonal variation present within subclones ofthe human metastatic melanoma line RVH421 to investigate interrelationships between cellular responses, in particular DNA replicationand DNA repair, following exposure to the model DNA damagingagent, UV-C. Although our UV-resistant lines possessed damage-

resistant DNA and RNA synthesis, it was the finding of enhancedmelanoma DNA repair that we interpret to underlie the primaryresistant phenotype. We propose that loss of other functions, includingthose which are expressed as defective PRR, as may be the case forsubclones R13 and R16, can result in the generation of sensitivevariants from a resistant background.

The 2-fold enhancement of UV resistance we report for RVH421,

a selection of its subclones and the independent melanoma line FM, iscomparable to that reported by some authors (11, 38), although notquite as extreme as others (10). Nonexaggerated resistance has beenreported in one instance (39). It is pertinent to note that melanomalines derived from xeroderma pigmentosum patients have been reported to be as UV sensitive as XP fibroblasts, presumably due to theirgross defect in nucleotide excision repair (40, 41).

We tested and eliminated the possibility that the degree of mela-

nization, or photoprotection, underlies the observed differences in UVresistance by the finding of initial dimer yields in our melanoma linesthat were equivalent to the EJ30 control. It is clear that in vivo,epidermal melanocytes and the melanins they produce act as a majorprotection system to limit photochemical damage to the skin (42).However, the current consensus is that melanins may not protect DNA

from direct photochemical damage in vitro. For example, measurements of CPDs in melanotic and amelanotic cells irradiated withUV-B or UV-C reveal similar levels of damage (11, 43) and no UV

survival advantage has been reported in cases where the effect ofdifferences in degree of melanization has been tested (10, 39, 44).

We demonstrated that the UV-resistant lines RVH421 and RIO

possessed supranormal kinetics of recovery of DNA and RNA synthesis following UV irradiation. Coexpression of UV-resistant mela

noma DNA synthesis and survival has been inferred previously bycounting the numbers of heavily labeled nuclei in cells that were given[3H]thymidine after UV irradiation (9), although melanoma RNA

synthesis recovery, normal or otherwise, does not appear to have beenreported previously.

Both PRR and NER processes have been implicated in the recoveryof DNA and RNA synthesis after irradiation (29, 31, 32), although itis believed that repair of DNA is not preferentially targeted to activelyreplicating sequences (45). It has been suggested that selective repairof transcribed regions of the genome may permit expression of genescoding for recovery functions (46), although it is also possible thatPRR processes themselves, by permitting replication of a damaged coding sequence, are a legitimate means of restoring transcription from thatsequence by producing a new undamaged coding strand (47).

In this report we have shown, by two independent means, thatalthough all of our cell lines possessed equivalent rates of replicÃ³njoining in the absence of DNA damage, UV-resistant melanoma linesdisplayed a 2-fold enhancement of UV-induced PRR in comparison to

melanoma subclones and a human tumor control, EJ30, possessinglower UV resistance. The enhanced PRR was also superior to thatreported by other authors for wild-type mammalian cells (19, 48).

This strongly suggests that a link exists between improved PRR andthe acquisition of UV resistance.

Based on evidence of UV-resistant DNA synthesis, Lavin et al. (9)suggested that human melanomas owed their resistance to the cyto-toxic effects of UV to a highly efficient "postreplication repair system", although this was not formally demonstrated. That PRR was

indeed enhanced in several murine melanoma lines was shown by Hilland Setlow (13), whose alkaline sucrose gradients revealed a 2-fold

increase in PRR rate. However, although suggested, the question of acorrelation between PRR and UV resistance was not formally addressed (13). In addition, the data of Konishi (11) reveal that his UVresistant melanoma line MIMI showed a 2.1-fold greater rate of PRR

compared to a HeLa control. Thus one can identify a consistentpattern into which our UV-resistant melanoma cell lines fit, of adoubling in the rate of UV-induced melanoma PRR.

JOO

6 12 18Time (hrs post irradiation)

24

Fig. 9. Rapid removal of antibody binding sites associated with ToT CPDs inmelanoma cell lines. Cell irradiation and experimental protocol were as described in Fig.8B. RVH421 (â€¢),RIO (d), R13 (O), EJ30 (A), FM (A), A375 (+), and HeLa (x) cellswere irradiated with 10 J/m2. Points, means of two to three determinations in duplicate;

bars, SEM.

187




PRR experiments can only be correctly interpreted if the rates ofDNA repair in the lines being compared are also established. Ourincision experiments revealed an unusual initial accumulation ofUV-induced DNA breaks in the melanoma cells, compared to the

controls EJ30 and HeLa. The break accumulation data of these controls are the same as that given for a variety of nonmelanoma humantumor lines by Squires et al. (22). Thus, we believe the melanomacells possess a previously unsuspected enhanced incision capacity.This was corroborated by immunoassays of (6-4) and CPD repair,

which indicated enhanced removal of both lesions by all our melanoma lines. Comparison with previous reports of human cell repaircapabilities (37, 49, 50) suggests that the melanomas accomplishrepair with an efficiency that lies at the extreme end of the range forhuman cells. In contrast, the incomplete repair of CPDs by HeLa andEJ30 mirrors the low levels of repair of inactive loci reported innumerous Bohr-Hanawalt assays of wild-type human cell repair (e.g.,

see Ref. 51). Earlier studies did not note enhanced repair by melanoma cells, but involved less optimal means of measurement, involving unscheduled DNA synthesis and thin layer chromatography afterhigh UV-C exposure (10, 11).

Thus our combined repair data provide the first evidence of anoverall elevation of photoproduct repair in melanoma cells, whichdoes not relate to the differences in UV survival between the variouslines, but may explain the generation of resistance per se. The 2-foldenhancement of early loss of (6-4)s and CPDs may well account forthe exaggerated rates of PRR in the UV-resistant lines. This is because

fewer lesions will be left in the template to interrupt replication duringthe chase period (which coincides with enhanced repair).

The lack of apparent PRR enhancement in the R13/R16 class ofmelanomas suggests they may have lost a PRR function, and onlyshow near-normal levels of UV resistance due to their enhanced repair

capabilities. Loss of a critical function in R13 is supported by therecessive nature of its UV sensitivity in hybrids, and that this may bea PRR function is suggested by the finding that R13 cells are moresensitive than the UV-resistant melanoma clones to killing by 6-thio-guanine5 which is thought to kill cells though S-phase-dependent

processes (52). Alternatively, or in addition, the targeting of repair atthe gene-specific level might account for differences in UV sensitivity

of the various clones; because only a few percent of the genome aretranscriptionally active, preferential repair (transcription coupled) ismasked by repair of the rest of the genome (53).

The genetic lability of tumor cells, as expounded by Nowell (54),may explain the origin of the differences within the RVH family ofsubclones and accords with the finding of widely varying properties ofsubpopulations of cells within tumor samples (55). Such variation hasbeen shown to be present within melanomas of recent origin, and notan artifact of continuous cell culture in vitro (56) and provides a poolfrom which metastatic variants arise (57, 58). Subpopulations of cellsresistant to DNA damaging agents, when already present within atumor on presentation (2), may play a pivotal role in repopulation ofthe tumor when placed under selection by chemotherapy. Such potential for clonal evolution during chemotherapy has been demonstrated in human gliomas (59) which, like melanomas, continue todefy clinical treatment.

Clinically relevant levels of resistance to platinum-based com

pounds appear to vary by only severalfold above the norm (60). Agrowing number of studies now confirm the significance of DNArepair in conferring such levels of cisplatin resistance to a variety ofhuman tumor cell lines (61-64). Fitting into this picture, our UV-

resistant melanoma lines were found to be coresistant to cisplatin, andwe note that there are several instances of UV-sensitive mammaliancells showing cross-sensitivity to cisplatin (65). This association be

tween cisplatin and UV sensitivity may be attributable to the appli

cation of common elements of the NER pathways to repair of helix-

distorting lesions induced by both agents.Finally, we feel that melanoma cells may well inherit their en

hanced repair capabilities (of UV-induced and, presumably, cisplatin-

induced lesions) from their melanocyte progenitors, which are regularly subjected to environmental insult in vivo. Very few data existconcerning melanocyte repair (43) although there are indications ofmelanocyte resistance to killing by UV treatment (44, 66, 67). Webelieve that repair proficiency, among other factors, may play acentral role in permitting the melanocyte to perform its protectivefunction in the epidermis and have serious consequences for therapyif the cell undergoes malignant transformation. However, the intrinsicgenetic lability of melanoma cells may result in loss of functions,including those required for damage tolerance, thus creating a class ofless resistant cells more amenable to therapy. The molecular origins ofmelanoma repair proficiency remain a future goal but may be predicted to involve the p53 pathway which commonly remains intact incutaneous melanoma cells (68), may be particularly damage inducible(69), and may mediate replication/repair events via proliferating cellnuclear antigen (69, 70).

ACKNOWLEDGMENTS

We should like to thank Dr. R. Kelsell, Dr. S. Squires, and J. Coates forhelpful discussions and reading of the manuscript and R. Northfield forexcellent technical assistance.

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1995;55:181-189. Cancer Res David H. Hatton, David L. Mitchell, Paul T. Strickland, et al. CellsDamage-Resistance Phenotype of Human Malignant Melanoma Enhanced Photoproduct Repair: Its Role in the DNA

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