MOLECULAR AND CELLULAR BIOLOGY, Sept. 1988, p. 3964-3968 Vol. 8, No. 90270-7306/88/093964-05$02.00/0Copyright C 1988, American Society for Microbiology

A Reduced Rate of Bulky DNA Adduct Removal Is Coincident withDifferentiation of Human Neuroblastoma Cells Induced by

Nerve Growth FactorLEE JENSENt AND STUART LINN*

Department ofBiochemistry and Group in Biophysics, University of California, Berkeley, Berkeley, California 94720

Received 18 February 1988/Accepted 14 June 1988

Human SY5Y neuroblastoma cells which were differentiated in culture by treatment with 7S murine nervegrowth factor for 5 weeks and selection with aphidicolin (L. Jensen, Dev. Biol. 120:56-64, 1987) demonstrateda considerably slower rate of removal of DNA adducts of benzo[a]pyrene, benzo[a]pyrenediolepoxide, andN7-methylguanine than did undifferentiated mitotic cells. A dramatic decline in unscheduled DNA synthesisinduced by UV radiation was similarly observed. DNA polymerase I and uracil DNA glycosylase wereunchanged after differentiation, DNA polymerase a and DNA methylase decreased roughly threefold, and totalapurinic-apyrimidinic endonuclease activity increased roughly threefold after treatment.

Mature neurons are terminally differentiated to a nonmi-totic state, and adult mammals do not have a pool ofneuroblasts from which to replace dead or impaired neuronalcells. Thus, it would be expected that the extent to whichneurons are capable of correcting DNA damage would affectthe survival of individual cells and ultimately the entirenervous system. It has not been feasible to obtain maturemammalian neuronal cells in sufficiently large numbers toinvestigate their DNA repair properties because they arenonmitotic and complexed tenaciously to many other typesof cells. Nonetheless, Lieberman and Forbes (22) observedthat neurons in rabbit cerebellar slices did not exhibitunscheduled DNA synthesis (UDS) in response to UV light,whereas glial cells in the same slices did. Likewise, usingdeveloping hamster brain slices, Gensler (5) found that UDSin response to UV light appeared to be lost at about the timethat those neurons became mitotically inactive, while Kar-ran et al. (16) reported a 10-fold reduction in repair DNAsynthesis induced by methyl methosulfonate or N-acetylaminofluorene between days 6 and 15 of embryogene-sis in neural retinal cells. Unfortunately, it cannot be con-cluded from such studies of repair DNA synthesis whether areduced capacity for DNA repair is being observed orwhether DNA repair is accomplished via an alternativepathway, utilizing, for example, shorter patches of synthe-SIS.We have investigated further the DNA repair properties of

neurons by using as a model system the clonal human SYSYneuroblastoma cell line. SY5Y neuroblastoma cells can beinduced to differentiate by culturing over a period of 5 weeksin medium supplemented with 7S nerve growth factor (NGF)(Fig. 1). Aphidicolin also is added during week 2 of NGFtreatment to select for cells which have responded mostrapidly to NGF. As a consequence of this regime, theneuroblastoma cells acquire many properties indicative ofmature peripheral neurons: they enter a terminally nonmito-tic state, produce long neurite bundles, develop significantresting potentials, require the continued presence of NGF

* Corresponding author.t Present address: Department of Neurobiology, Stanford Uni-

versity School of Medicine, Stanford, CA 94305-5401.

for survival, and stain intensely for intracellular monoamines(15).When naive neuroblastoma cells (those not exposed to

NGF) and NGF-differentiated cells were irradiated with254-nm light for various times at a constant dose, labeledwith [3H]thymidine, and examined by autoradiography,markedly fewer grains were noted over the nuclei of theNGF-differentiated cells than over the nuclei of the non-S-phase naive cells (Fig. 2). The NGF-treated cells incorpo-rated very little label even after an abnormally long, 12-h,labeling period. In contrast, the naive cells exhibited adose-dependent response to UV light, and label incorpora-tion was significantly higher at the longer incubation time.Thus, the cells appeared to lose the ability to exhibit UDS inresponse to UV irradiation after differentiation in culture ina fashion comparable to that reported for neuronal cells inanimal brain slices (5, 22).The primary class of lesions produced in DNA by UV

irradiation is the cyclobutane dimer (32), which is repairedvia the nucleotide excision repair pathway used for mostforms of bulky-lesion repair (10, 40). To assay directly forthe excision of bulky lesions, we exposed cells to[3H]benzo[a]pyrene for 18 h and harvested them at varioustimes thereafter. The rate of disappearance of adducts of[3H]benzo[a]pyrene from the DNA of NGF-differentiatedcells was significantly slower than that from the DNA ofnaive cells (Fig. 3A); the naive cells removed adducts with ahalf-life of approximately 2 days, whereas the NGF-differ-entiated cells removed adducts with a half-life of approxi-mately 12 days. In the latter case, significant and easilydetectable amounts of adducts remained after 30 days.The above-described results could be equivocal for sev-

eral reasons. Since benzo[a]pyrene requires metabolic acti-vation by the cell, it is not possible to pinpoint the true endof the incubation period with the agent; i.e., it is not knownhow much unmetabolized benzo[a]pyrene might remain inthe cell to be activated at later times. Another problem isthat the rates and extents of formation as well as thedistribution of the active benzo[a]pyrene metabolites mightdiffer for the two cell types. Therefore, the experiment wasrepeated with the activated derivative (+)-r-7,t-8-dihydroxy-t- 9,10- epoxy - 7,8,9,10- tetrahydrobenzo[a]pyrene - 1,3 - [3H](anti) (3H-BPDE). Cells were exposed to 0.1 or 0.8 ,uM

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FIG. 1. Human SY5Y neuroblastoma cells. (A) Untreated(naive) cultures. (B) Cultures exposed to 7S murine NGF for 5weeks and selected during week 2 with aphidicolin as describedpreviously (15). Bars, 100 ,uM.

3H-BPDE for 1 h, and the DNA was isolated from nuclei atvarious times thereafter. The results were similar to thosefound with benzo[a]pyrene: the half-lives were approxi-mately 2 and 22 days for naive and NGF-differentiated cells,respectively, at the lower dose; at the higher dose thehalf-life for naive cells was 4 days, and there was nodetectable removal in NGF-differentiated cells (Fig. 3B).

It should be noted that whereas the rate of bulky-adductremoval was reduced at least sixfold for the differentiatedcells, increasing the labeling period sixfold (from 2 to 12 h)after UV exposure did not result in detectable UDS. Thus,the lack of detectable UDS in response to UV light might notbe totally accounted for by the reduced rate of adductremoval.

Cells were also exposed to 10 ,uM 3H-methyl-N-nitro-sourea, and DNA was isolated from nuclei at various timesafter exposure, hydrolyzed, and analyzed. N7-Methylgua-nine (N7-MeG) was the only adduct that could be detected ineither naive or NGF-differentiated cells, and the rate ofremoval of this adduct was faster in the naive cells (half-lifenear 35 h) than in the differentiated cells (half-life, near 104h) (Fig. 4). The latter value approximates the estimatedthermal half-life of N7-MeG in DNA (96 to 102 h at 37°C[34]). In mammalian cells, it has not been firmly establishedwhether N7-MeG is removed by nucleotide or base excisionrepair. The reduction in the repair of this lesion after NGFtreatment might suggest that N7-MeG is repaired by nucle-

aia

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10 20 30 40 50 60 70 80 >90&rns/Nudko

10 20 30 40 50 60GMnsNudew

70 80 >90

0 5 10 15 20Dose (J/m 2 )

FIG. 2. Effect of UV light on UDS. UDS was induced byexposing cells grown on 60-cm2 petri dishes (Falcon) to 254-nm UVlight at a dose of 0.5 J/m2 and then labeling them in completemedium containing 10 pCi of [3H]thymidine per ml for either 2 or 12h. Cultures were then washed in Dulbecco modified Eagle medium(DME) and incubated in unlabeled medium for 30 min. The cellswere fixed to the plastic with methanol-acetic acid (3:1), washedwith 70%o ethanol, and then flushed with water. Air-dried plates werecoated with Nuclear Track Emulsion (NTB3; Eastman Kodak Co.)and exposed for 7 days at 4°C prior to development. The number ofexposed grains above each nucleus was determined microscopicallyfor non-S-phase cells. (A) 5 J/m2, 12-h labeling period. (B) 20 J/m2,12-h labeling period. Solid bars, Naive cells; hatched bars, differen-tiated cells. (C) Dose response to UV light. Symbols: l, naive cells,2-h labeling period; O, naive cells, 12-h labeling period; *, NGF-differentiated cells, 2-h labeling period; *, NGF-differentiated cells,12-h labeling period.

B

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100

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DaysFIG. 3. Removal of bulky DNA adducts. For mitotic cl

cultures were prelabeled for 2 days with 0.1 ,uCi of [(4C]thymiper ml to assess and correct for the extent of cell division.cultures were mixed and replated several days prior to the addiof a carcinogen to obtain uniformly prelabeled cultures. Ntreated cells were not prelabeled, as they were not mitotically ac(15). In the case of BPDE treatment, parallel cultures of cells v

prelabeled with [3H]thymidine as described above and exposeiunlabeled BPDE to assess mitotic activity during the experim(A) Cells ( 3 x 107 to 8 x 107 per 150-cm2 flask) were exposed,uM [3H]benzo[a]pyrene (70 Ci/mmol; New England Nuclear Ccfor 18 h at 36°C in a 5% CO2 atmosphere, washed three times i

warm DME, harvested at the times indicated after exposure, frcin liquid nitrogen, and stored at -80°C. The frozen samples v

thawed and suspended at 0°C in 10 mM Tris hydrochloride (pH E5 mM EDTA at 108 cells per ml, and lysed in 10 volumes of 0.

EDTA (pH 8.0)-0.5% sarcosyl-150 p.g of proteinase K by incubaat 50°C for 3 h. The DNA was extracted twice with 2 volumebuffer-saturated phenol-chloroform-isoamyl alcohol (25 :24: 1).aqueous phase was extracted with 2 volumes of chloroform-isoaalcohol (24:1), dialyzed, and incubated with boiled RNase A,ug/ml) for 3 h at 37°C. The DNA was dialyzed extensively agaim,mM Tris hydrochloride (pH 7.5)-10 mM MgCl2, and derivativesDNA were quantitated by measuring the radiolabel present an

otide excision repair in these cells, in agreement with indi-cations that xeroderma pigmentosum cells, which are defec-tive in the removal of bulky adducts, also remove N7-MeGmore slowly than do normal cells (35). Other aklylatedpurines which can be detected in isolated calf thymus DNAtreated with 10 ,uM 3H-methyl-N-nitrosourea (a barely toxiclevel) were not detected in samples of DNA obtained fromneuroblastoma cells in either state of differentiation. Similarresults reported for a number of cell types (26, 33, 37) have

^ been taken to indicate very rapid repair rates for theseadducts.

Several DNA glycosylases, apurinic-apyrimidinic (AP)endonucleases, and DNA polymerases have been describedin detail and shown to participate in mammalian DNA repair.

30 Activity levels of several of these enzymes were assayed toascertain whether changes would occur as a consequence ofNGF exposure (Table 1). DNA polymerases were isolatedby two DEAE-cellulose chromatographic steps and assayedwith "activated" salmon sperm DNA (17, 18, 31). Theywere identified by their chromatographic behavior (2, 4, 8, 9,29) and by their sensitivity to aphidicolin, N-ethylmaleimide,or 2',3'-dideoxythymidine (1, 12, 14, 38). AP endonucleaseswere assayed with depurinated phage PM2 3H-DNA as thesubstrate (28). Two peaks of activity separated upon phos-phocellulose chromatography: one with a 10 mM potassiumphosphate wash and another at 225 mM potassium phos-phate in a linear elution gradient (correlating almost pre-cisely to AP endonucleases I and II, respectively, fromcultured human fibroblast cells). Uracil DNA glycosylasewas assayed with phage PBS 3H-DNA as the substrate (20).DNA methylase reactions (100 ,ul) contained 100 mM imid-azole-NaOH (pH 6.5), 0.5 mM dithiothreitol, 20 mM EDTA(pH 6.5), 25 ,uM S-[methyl-3H]adenosylmethionine (1 Ci/mmol), and 0.53 mM Micrococcus luteus DNA. After 20 minat 37°C, samples were processed as described previously(21). All values in Table 1 represent rates within the linearresponse range. Cell counts were determined at the time ofharvest with a hemacytometer.DNA polymerase a decreased about threefold, while

DNA polymerase Pi remained relatively constant before andafter differentiation; these results were similar to those foundfor comparisons between other mitotic and nonmitotic pop-

1 2 ulations of cells (13, 19). Total AP endonuclease activityincreased approximately threefold; approximately 20% of

ells, each sample chromatographed with AP endonuclease I fromdine cultured human fibroblast cells. Uracil DNA glycosylaseThe remained nearly constant, while DNA methylase decreasedition to approximately 38% the level in naive cells. In sum, baseIGF-tivevered tolent.to 1)rp.)withDzenvere3.0)-.5 MLtions ofTheLmyl(100st 10andd by

fluorometric procedures (11, 36), respectively. Symbols: El, naivecells; *, NGF-differentiated cells. (B) Cultures were exposed to 0.1or 0.8 ,uM 3H-BPDE (1.38 Ci/mmol; Chemical Carcinogen Refer-ence Standard Repository, National Cancer Institute) for 1 h inEarle salts at 36°C, after which samples were harvested at the timesindicated as described above. DNA was isolated (23) from nuclei(25). Frozen nuclei were thawed in 1 ml of 6 M guanidine hydro-chloride-10 mM EDTA (pH 7.0) and sonicated. Unbound hydrocar-bon was removed by extracting the lysate extensively with ethylacetate. The lysate was brought to 6 M guanidine hydrochloride, andDNA was purified by equilibrium centrifugation in 2.2 M cesiumsulfate-10 mM EDTA (pH 7.0)-9% dimethyl sulfoxide. DNA fromindividual fractions was ethanol precipitated and suspended in 10mM Tris hydrochloride (pH 7.5). After the DNA concentration was

determined, the DNA was collected by acid precipitation onto glassfiber filters, and the 3H content was quantitated. Symbols: O, and O,naive cells; * and *, NGF-differentiated cells; O, and *, 0.1 ,uM3H-BPDE; O and *, 0.8 ,uM 3HBPDE.

IE

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10000cSa

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FIG.Cells weNew EnCO2. Nu

tide excision occurs at a significantly reduced rate, if at all.Unrepaired damage to bulky DNA might ultimately manifestitself at the transcriptional level, as lesions induced by UVand BPDE have been shown to inhibit transcription (7, 30).Recent reports have shown that transcriptionally activeregions of DNA are repaired more rapidly than are nontran-scribed regions (24, 27); perhaps in nonmitotic cells nucleo-tide excision repair is manifested only upon active geneswhich represent such a small fraction of the total DNA thatsuch repair would be undetectable by standard procedures.Such selective repair would avoid unnecessary disruption ofinactive chromatin and the problems inherent in reestablish-ing the complex chromatin structure, and chromosomalproteins have been reported to reduce excision repair in

0 2 4 6 8 1 0 mammalian cells (3, 39). Certain DNA adducts might beDMys tolerated to some degree in nontranscribed regions, yet an

accumulation of such adducts might eventually alter chro-4. Removal of 3H-methyl-N-nitrosourea DNA adducts. matin structure and ultimately affect DNA function. Such anre exposed to 10 ,uM 3H-methyl-N-nitrosourea (1 Ci/mmol; accumulation of damage within neuronal cells would haveigland Nuclear Corp.) for 50 min in Earle salts at 36°C in 5% unique physiological consequences, as dysfunctional cellsiclei were isolated, lysed in 1 ml of 0.5% sarcosyl, extracted would not be replaced by mitotic activity.ith% I ()A|n1vnf wouldrn_iantnothsbe> relae by mioi activity.twIce wiln z volumes Oi cnioroiioiUm-isoamyl alIconoi kL': i), Ulalyzea

against 10 mM Tris hydrochloride (pH 8.2), incubated with 200 1Lg ofboiled RNase A per ml for 15 min followed by 150 ,ug of proteinaseK per ml for 1 h at 37°C, and finally dialyzed against 10 mM Trishydrochloride (pH 8.2) overnight. DNA was hydrolyzed in 0.1 NHCI in sealed glass ampoules at 70°C for 45 min, and hydrolysateswere neutralized with 5 M potassium hydrophosphate, filteredthrough YMT filters (Amicon Corp.), applied to a Supelco LC-18column (4.6 mm [inner diameter] by 25 cm), and analyzed byhigh-performance liquid chromatography. After injection, the col-umn was washed for 3 min with 50 mM potassium phosphate (pH7.6) at a flow rate of 1 mnmin. A linear gradient of elution bufferfrom 50 mM potassium phosphate (pH 7.6) to the same buffercontaining 3% acetonitrile was applied to the column over a periodof 45 min at a flow rate of 1 mllmin. Fractions were collected directlyinto scintillation vials and counted in 4.5 ml of Scint A (PackardInstrument Co., Inc.). Identification of N7-MeG was based uponcomigration with an internal standard. Results were normalized tothe area of the adenine absorbance peak. Symbols: I, naive cells;U, NGF-differentiated cells. O.D. 259, Optical density at 259 nm.

excision repair enzymes would appear not to be reduced byNGF treatment-the effects upon uracil DNA glycosylaseand AP endonuclease activities were of the same order asthose noted when cultured mouse fibroblasts became non-mitotic at late passage (20). Interestingly, those cells alsoappeared to have little or no nucleotide excision repair.Mammalian neurons are terminally differentiated at birth,

so DNA repair must play a critical role in maintaining thefunctional capabilities of these cells throughout the life of theorganism (6). The results of this study imply that while baseexcision may be normally functional in these cells, nucleo-

TABLE 1. Enzyme activities in SYSY cells prior to and afterexposure to NGF

U/106 cellsEnzyme

Naive NGF differentiated

DNA polymerase a 2.8 0.8DNA polymerase ,3 0.15 0.16AP endonuclease

I 7.5 18.1Total 32.6 90.7

Uracil DNA glycosylase 30.8 25.6DNA methylase 3.2 1.2

We thank Sharon Krauss, Michael LaBelle, Mary Hitt, and DaleMosbaugh for technical advice, Ronda Ching for editorial assis-tance, Cornelius Tobias for helpful discussions during the initialstages of the project, and Eric Shooter for use of his facilities.

This work was funded by grant 76EV70190 from the U.S. Depart-ment of Energy and by Public Health Service training grantCA09272 from the National Cancer Institute.

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