Proc. NatL Acad. Sci. USAVol. 78, No. 6, pp. 3654-3658,. June 1981Cell Biology

Correlation of double-minute chromosomes with unstablemultidrug cross-resistance in uptake mutants ofneuroblastoma cells

(cancer/maytansine/vincristine/Baker's antifol/adriamycin)

FRED BASKIN*, ROGER N. ROSENBERG*, AND VAITHILINGHAM DEVtDepartments of *Neurology and tPathology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235

Communicated by Robert T. Schimke, March 2, 1981

ABSTRACT A series of increasingly drug-resistant cell pop-ulations were selected and cloned from C-46 murine neuroblas-toma with the chemotherapeutic drugs maytansine, vincristine,adriamycin, or Baker's antifol. All clones demonstrated reciprocalcross-resistance to these structurally and functionally diversedrugs and failed to accumulate radiolabeled vincristine, colchi-cine, or Baker's antifol despite normal drug binding to cell ho-mogenates. Initial isolates of drug-resistant populations were ge-netically unstable, rapidly reverting to a drug-sensitive phenotypewhen grown without drug, at 0.05 reversion per cell division. Afterprolonged growth in drug, this drug-resistant genotype stabilized.Mean chromosome number increased 300% in an initially isolated20-fold maytansine-resistant clone, which also displayed numer-ous double-minute chromosomes. Descendants 240-fold more re-sistant than the parent, also unstable, possessed the wild-type com-plement of 80 chromosomes, but 45% of these cells possessed 24double-minute chromosomes per cell; such chromosomes wereabsent from the drug-sensitive parental clone. Only 1.0 and 1.2double-minute chromosomes per cell were seen in a 7-fold stablyresistant revertant or 1200-fold stably resistant descendants, re-spectively. Double-minute chromosomes containing amplifiedgenes for the drug target dihydrofolate reductase (tetrahydrofo-late dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreduc-tase, EC 1.5.1.3) have been reported in an unstable methotrexate-resistant Ri-A sarcoma. These extrachromosomal gene copieswere absent in stably resistant progeny. The presence of similarparticles in unstably drug-resistant uptake mutants of neuroblas-toma and their diminution in stably resistant descendants supportsand extends their possible role in the rapid onset and instabilityof epigenetic drug resistance in cancer chemotherapy.

We have previously reported "epigenetic" mutants of neuro-blastoma cells resistant to 5-fluorodeoxyuridine because of am-plified production of the parental drug-specific enzyme, thy-midylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) (1-3). We could not then ex-plain the initial marked instability of this phenotype when thecells were grown in the absence ofdrug or the subsequent emer-gence ofa stably resistant phenotype after further growth at theselective drug concentration. Kaufman et al. (4) have since de-scribed similar methotrexate-resistant murine sarcoma cells inwhich the unstably resistant phase was correlated with the pres-ence ofdouble-minute chromosomal spheres (DMS) containingamplified gene sequences for the increased production of thetarget enzyme, dihydrofolate reductase (tetrahydrofolate de-hydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase,EC 1.5.1.3). These were greatly reduced in subsequently iso-lated stably resistant descendants, suggesting a causal relation-

ship between DMS and epigenetic drug resistance. In an effortto support and extend this hypothesis, we contrasted the chro-mosomal profiles of unstable 20- and 240-fold maytansine (MT)-resistant populations with their stably 1200-fold resistant prog-eny and a drug-sensitive revertant.

All previously characterized unstably drug-resistant or am-plified-gene mutants display increased levels of drug-bindingcytoplasmic "target" enzymes (1-11). This type of drug-resis-tance mechanism was unlikely for the tubulin- and DNA-bind-ing drugs emphasized here. Therefore, we have also contrastedthe abilities of our growing mutants to accumulate the drugsused in their selection with the abilities of their cell-free ho-mogenates to bind these drugs.

MATERIALS AND METHODSCell Lines. Clone C46 of the C1300 mouse neuroblastoma

cell line was a gift of Gordon H. Sato (University of California,San Diego). Cell monolayers were routinely subcultured andgrown as described (1-3). A line resistant to the mitotic spindle-disrupting drug MT was derived by exposing rapidly dividingcells to MT at 1.0 ng/ml (12) in Dulbecco's modified Eagle'smedium (GIBCO) supplemented with 10% fetal calf serum(Reheis Chemical, Kankakee, IL). Most of the cells showed cy-totoxic effects and began floating offthe substratum after 3 days.Two weeks (approximately 10 cell generations) after the initialexposure to drug, small colonies ofviable cells began to appear.Several colonies were individually dispersed by trypsinizationand diluted, and the cells were allowed to grow into clonal col-onies. One of these clones, C-46 MT-1, was propagated andstored under liquid nitrogen for later study. One flask was sub-cultured into medium containing drug at 10 ng/ml, and theselection process was repeated, yielding C-46-MT-2. Eventu-ally a clone of cells that grew rapidly in 50 ng/ml was estab-lished, C-46-MT-3. All three resistant cell lines are morpho-logically very similar to the cell line from which they werederived; that is, under phase-contrast microscopy the cells ap-pear dark, square, and without neurites. C-46-MT-2 cells werealso subcultured and grown without MT for 4 months, at whichtime it was, determined that this population was unable to growat the original selective drug concentration, 10.0 ng/ml. Wedesignate this revertant line C-46-MT-2-R. The generationtimes of the drug-sensitive, the three resistant, and the rever-tant variants are 20, 28, 24, 38, and 20 hr, respectively, at 370C.Other populations- of cells resistant to vincristine (VC), adria-mycin (ADR), or Baker's antifol (BAF) (13) were similarly se-lected and maintained for 6-12 months at their selective drugconcentration; they are designated C-46-VC, C-46-ADR, andC-46-BAF, respectively.

Abbreviations: DMS, double-minute chromosomal spheres; MT, may-tansine; ADR, adriamycin; BAF, Baker's antifol; VC, vincristine.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.

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Drugs. Chartreusin (NSC-5159), MT (NSC-153858), unla-beled and tritium-labeled VC (NSC-67574), unlabeled and 14C-labeled BAF (NSC-139105), and ADR (doxorubicin, NSC-123127) were supplied by the Division of Cancer Treatment,National Cancer Institute (Bethesda, MD). [3H]Daunorubicinand [3H]acetylcolchicine were purchased from New EnglandNuclear. 2,4-Dinitrophenol was purchased from Sigma.

Determination of Resistance (ECs5 Values). Resistance todrug was quantitated in terms of EC,% values (1-3). A series of10-14 Linbro four-well, 60-mm cluster tissue culture disheswere inoculated with 5 x 105 cells per well. On day 2, 1:2 di-lutions ofdrug were added (four wells per concentration) exceptfor four control wells without drug. After 3 days the cells weretrypsinized and counted with a Coulter counter (ZBI). The av-erage cell number at each drug concentration, expressed as apercentage of control, was then plotted against the drug con-centration on a logarithmic scale to determine the concentrationof drug effective in inhibiting the growth by 50% (EC50).

Determination of Stability of Resistance to Growth Inhibi-tion in the Absence of Drug. Cells from resistant populationsthat had been maintained in the drug concentrations used toselect them for either 2 weeks (all MT-resistant lines) or 6-12months (all cell lines) were subcultured into flasks containingnormal medium without drug. At 2-week intervals for 10 weeks(70 generations) these cells were subcultured and their degreeof resistance to growth inhibition was determined coordinatelyas described above.Chromosome Analysis. Logarithmically dividing cultures

were treated with Colcemid at a concentration of 0.1 ,ug/ml ofmedium for 30 min at 37°C. The cells were collected by mildtrypsinization followed by centrifugation. The pelleted cellswere suspended in hypotonic 0.075 M KCl solution for 7 minat 230C, centrifuged, and suspended in methanol/acetic acid(3:1, vol/vol) fixative for 1 hr. The fixative was changed twiceand the cells were suspended in 0.5 ml of this solution. Twodrops of this cell suspension were dropped onto cold wet slidesand allowed to dry at 23°C. The slides were stained with a 4%Giemsa stain and a minimum of20 well-spread metaphases werescored for the numbers of normal chromosomes and DMS.

Retention of Radioactively Labeled Drugs. Cells in expo-nential growth were plated on 60-mm Falcon cell culture dishes(1 X 106 cells per plate). On the third day two plates were re-moved and their cells were trypsinized and counted. Identicalplates were rinsed with Tyrode's solution (8 g of NaCl, 0.26 gof KCl, 0.2 g of CaC12, 0.1 g of MgCl26H2O, 0.05 g ofNaH2PO4 H2O, and 1 g of glucose per liter, pH 7.2) at 37°C.Uptake was then initated by adding 5 ml of Tyrode's solution

containing 1 puCi (1-10 tLM) of [3H]VC or [3H]acetylcolchicine,or 0.02 /iCi (1040 ,uM) of '4C-labeled Baker's antifol (1 Ci =3.7 X 101" becquerels). At various times between 30 sec and90 min the process was halted by aspirating the labeled solution,cooling the plates on ice, and rinsing the plates three times with10 ml of cold Tyrode's solution containing 100 /iM unlabeleddrug, taking care not to dislodge cells with each rinse. The cellswere then scraped and rinsed into distilled water, homoge-nized, and assayed for protein and radioactivity. All time pointmeasurements were done in triplicate and the samples con-tained at least 200 cpm over blanks, plates exposed to drug for30 sec at 0C. Drug retention was expressed as the ratio ofcpm/mg of protein per hr of drug-sensitive cells to that of resistantcells.Drug Binding and Dihydrofolate Reductase Assays of Cell

Homogenates. Specific binding assays of 1 X 105 dpm, 0.1 or2.5IM [3H]acetylcolchicine or 0.1 or 12 kM [3H]VC to tubulin-containing cell-free homogenates were performed with a mod-ification of the method of Sherline et al. (14). Aliquots of a freshcell-free sonicate (20 sec, Bronwell Biosonic IIA) were prein-cubated for 0-2.5 hr and then incubated with labeled drug for2.5 hr at 370C. Aliquots were mixed with 1.2 vol of activatedcharcoal in distilled water (4 mg/ml) at 0C for 10 min. Aftercentrifugation, supernatants were quantitated for radioactivity.Binding (less that ofa heat-inactivated control) was extrapolatedto a theoretical assay duration of 0 hr.

[3H]Daunorubicin (0.1 /Ci, 0.1 or 10.0 ,uM) was mixed withsonicate for 10 min at 230C. Bound drug was precipitated with20 vol of 5% trichloroacetic acid at 0C, the pellet was rinsedthree times and solubilized, and its radioactivity was measuredas above. All radioactive pellets contained at least 200 cpm overblanks containing only bovine serum albumin.

Dihydrofolate reductase was assayed spectrophotometricallyas described (15).

RESULTSReciprocal Cross-Resistance of Mutants. Cell lines initially

selected for their resistance to growth inhibition by the micro-tubule-disrupting drugs VC and MT (12), the anthracycline in-hibitor of polynucleotide synthesis ADR; or the dihydrofolatereductase inhibitor BAF (13) are all reciprocally cross-resistantto all four drugs (Table 1). Interestingly, in data not included,C-46-BAF was not cross-resistant to another dihydrofolate re-

ductase inhibitor, methotrexate. Again, these cell lines are notcross-resistant to chartreusin, another lipophilic cytotoxic drug.

Mutants Are Impaired in Their Ability To AccumulateDrugs in Vivo Despite Normal Binding of These Drugs to Cell

Table 1. Unstable drug resistance correlates with impaired drug retention and 10 times more DMS

Phenotypic Relative drug resistancet and (retention*)Cell line stability* MT ADR VC BAF Chromosomes DMS

C46(parental) S 1 1 1 (1) 1 (1) 77 ± 3 0C-46-MT-1 NS 20 4 18 (ND) 11 (ND) 320 ± 14 NDC-46-MT-2 NS 240 40 210 (50) 108(19) 80 ± 5 10.5 ± 8.2C-46-MT-3 S 1200 210 1050 (91) 550(52) 79 ± 5 1.2 ± 0.5C-46-MT-2-R S 7 3 5(1.1) 2 (ND) 80 ± 3 1.0 ± 0.2C-46-Vc S 30 12 20 (13) 20 (10) 71 ± 8 0C-46-ADR S 24 15 24(13) 20 (14) ND NDC-46-BAF S 8 10 14(10) 24 (21) 89 ± 12 0

Chromosome and DMS counts are given as mean ± SD. ND, not determined.* S, stable, drug resistance fell less than 10% per month of growth without selective drug; NS, not stable.t Ratio ofa given drug concentration required for 50% inhibition ofgrowth relative to that inhibiting the growth ofthe parentalC46 clone by 50%; data are the first numbers, without parentheses.

* Ratio ofcpm ofdrug retained per mg ofprotein per hr at 370C by parental drug-sensitive cell line to that ofthe resistant clones;data are the second numbers, enclosed in parentheses.

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Homogenates and Normal Dihydrofolate Reductase Activities.Net retention (influx less efflux) of radioactively labeled VC,acetylcolchicine, or BAF is impaired in all mutants tested, theimpairment at 1 hr being proportional to, but not stoichiometricwith, drug resistance (Table 1). Accumulations of these drugsincreased between 30 sec and 2 min and were linear after 5 min.In contrast to these intact cell experiments demonstrating con-sistent inhibition of net accumulation of drug, fresh cell-freehomogenates of all drug-sensitive and resistant cells boundidentical amounts of [3H]colchicine, [3H]VC, [3H]daunorubicin,and ["4C]BAF at both saturating and subsaturating drug con-centrations. These values suggest that the impaired accumu-lation of drugs seen in intact cells does not reflect fewer or al-tered drug-binding sites on tubulin, nucleic acids, or dihydrofolatereductase, respectively. Furthermore, the labilities of[3H]colchicine binding to homogenates of resistant and drug-sensitive cells, often cited as an index of structural differencesin the tubulin polypeptides of different organelles and tissues(16), were also identical, half-life = 3.8 hr. Consistent with the['4C]BAF binding data, the C-46-BAF line and its C-46 parentexpressed identical dihydrofolate reductase activities, 18 ,umoVmg of protein per hr at 37TC.

Addition ofunlabeled VC, ADR, or BAF at 200 ,u M increasedthe 30-min accumulation oflabeled VC by 350%, 250% and 80%respectively. Importantly, preincubation of resistant cells with1 mM dinitrophenol for 10 min enhanced the accumulation ofall labeled drugs.

Lability or Stability of Drug Resistance. The resistance togrowth inhibition of C46-VC, C-46-ADR, and C-46-BAF,available only after growth for 6-9 months in media containingdrug concentrations used in their selection, proved to be com-pletely stable upon extended growth in the absence of this se-lective pressure. In contrast to this, clonal variants of early andintermediate isolates of MT-resistant clones (C-46-MT-1 and -2), propagated in their selective MT concentrations for either2 weeks or 8 months, exponentially lost their drug resistancewhen grown without drug, with a logarithmic 50% reversioneach 10 and 12 days, respectively. It should be noted that al-though the drug resistance of C-46-MT-1 was completely lostafter only 8 weeks of growth without drug, a stable 7-fold com-ponent of the largely unstable 240-fold resistance of C-46-MT-2 remained in the C-46-MT-2-R revertant even after 12 monthsof further growth without drug. Interestingly, the drug resis-tance ofC46-MT-3, the more highly resistant progeny of C-46-MT-2, proved to be completely stable, that is, there was nodetectable reduction in drug resistance after 10 further monthsof growth without drug.

Chromosomal Examinations. Our C-46 parental drug-sen-sitive clone displays a stable heteroploid 77 chromosomes, in-cluding one small "marker" chromosome visible in most of ourdrug-resistant isolates. No DMS were seen in more than 40clear metaphases examined (see Table 1 and Fig. 1A). Giemsapreparations of our stable C-46-VC and C-46-BAF (see Fig. 1B)resistant variants were nearly identical. However, our series ofsuccessively isolated and increasingly MT-resistant variants dis-played consistent differences. C-46-MT-1 exhibited a majorchromosomal increase from its C-46 parent, a 300% increase to320 chromosomes (see Fig. 1C). DMS were seen in this prep-aration but were not scored because'of the difficulty in quan-titatively detecting them in these extremely crowded meta-phase spreads. C-46-MT-2, a more highly resistant clonaldescendant of C-46-MT-1, demonstrated a normal number ofchromosomes but displayed a mean of 10.5 DMS per cell (seeFig. 1D). These means do not reflect the considerable heter-ogeneity that exists in the distribution of DMS between indi-vidual cells. More specifically, 55% of these cells had no DMS,

the remaining 45% averaging 22. Two cells in 22 examined hadmore than 50; The 7-fold stably resistant revertant (C-46-MT-2-R) and the 1200-fold stably resistant progeny of this clone (C-46-MT-3) displayed normal chromosomal numbers and fewDMS (see Table 1, Fig. 1 E and F).

DISCUSSION

After experimental or clinical cancer chemotherapy, drug re-sistance commonly emerges with a frequency and rapidity dif-ficult to explain in terms of the relatively small fraction oftumorcells killed. Again, this initial resistance is often unstable, dis-appearing exponentially and rapidly with further growth of thecells without drug. After prolonged growth in drug, however,the resistant phenotype becomes stable. Because this drug re-sistance is a major barrier to the successful chemotherapeutictreatment of cancer, an understanding of how this resistance isinitiated and maintained is of considerable interest to both cli-nicians and scientists.

Kaufman et al. (4) reported the presence of large numbersof DMS in initial isolates ofunstably methotrexate-resistant Rl-A sarcoma cells. These contained amplified gene sequences al-lowing increased production of the target enzyme, dihydrofol-ate reductase. We previously described neuroblastoma mutantsin which resistance to 5-fluorodeoxyuridine and induced syn-thesis of thymidylate synthetase went through similar unstableand then stable drug resistance phases (1-3). We would like tostress that both in our laboratory and in the literature, unstablyresistant mutants, although often not characterized or discussed(17), are quite common. This instability is itself exact and sur-prisingly invariant. The remaining resistance plotted on a log-arithmic scale is inversely linear with cell doublings withoutdrug (figure 2 of ref. 1), approximately a constant-50% loss each10-12 days for 5-fluorodeoxyuridine (1), methotrexate (4, 5, 7),and MT.We are currently characterizing the biochemical mechanism

of resistance in a class of these unstably resistant neuroblastomamutants. We have documented a broad reciprocal cross-resis-tance pattern and an inability to accumulate structurally andfunctionally diverse drugs, despite their normal binding to cell-free homogenates. The cross-resistance seen between mitoticspindle (tubulin) and DNA-binding drugs in our mutants isquite common (18-31) and, where characterized, the mecha-nism of resistance frequently involves either a decreased per-meability of the cells to drugs that enter by passive diffusion(20, 21, 28) or an increased efflux activity extending to both drugclasses (22-26). Cross-resistance to, and inability to accumulate,a water-soluble antifolate such as BAF, and normal levels ofdihydrofolate reductase in such resistant cells, is much rarer(17). Our correlation of drug resistance with failure to accu-mulate these drugs (Table 1) clearly indicates that these cellsare uptake mutants. Enhanced uptake after dinitrophenol de-pletion ofATP pools is consistent with their being efflux mutants(22, 25, 26).

Our central observation of numerous DMS in our unstablyresistant C-46-MT-2 clone and their great diminution in our C-46-MT-2R revertant and C-46-MT-3 stably resistant progenysupports the hypothesis of Kaufman et al. (4) that amplifiedgene-containing DMS are responsible for the unstable (epige-netic) phase of drug resistance. Their appearance in our neu-roblastoma mutants extends their role to a drug-resistant neu-rological tumor cell line and to probable transport mutants. Inother celllines, their disappearance and the emergence ofstabledrug resistance is further correlated with the emergence of ho-mogeneously staining regions, which also contain amplifiedgene sequences (4, 5), but integrated into stably replicating

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FIG. 1. Giemsa-stained metaphase chromosome spreads of cells. (x630-1000.) (A) C-46 (parental); (B) C-46-BAF; (C) C-46-MT-1; (D) C-46-MT-2; (E) C-46-MT-2-R; and (F) C-46-MT-3. Note large numbers of DMS in C-46-MT-2, which is unstably resistant to MT.

chromosomes. Because homogeneously staining regions havebeen reported to occur frequently without drug selection indiploid lines of human neuroblastoma (32, 33), only their ab-sence in our C-46 parental line would allow an unambiguousinterpretation of this experiment.

In work not shown here, two-dimensional electrophoreticgels ofproteins from unstably MT-resistant cells containing nu-

merous DMS were contrasted with gels of proteins from drug-sensitive cells. No differences in some 200 ofthe most abundantproteins were visible. It may be possible to more sensitivelydetect the resistance-producing gene product by in vitro trans-lation of mRNA isolated by hybridization to double-minute

DNA (34). Although no specific gene product has yet beenshown to cause the membrane-dependent inhibition ofthe drugaccumulation pattern described here, the DMS seen in initialisolates of unstably MT-resistant cells could contain amplifiedgenes for a membrane phosphorylase, glycosyltransferase, or

glycosidase. This would explain the pleiotropic changes in theplasma membrane glycoconjugates seen in our and other (25-31) broadly cross-resistant cancer cell lines.

Double-minute chromosomes, most frequently seen inneural tumors (35), are variously believed to arise from (35), or

become integrated into (4, 5), homogeneously staining regions.In an effort to see if drug exposure itself produced DMS par-

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ticles, we exposed C-46 cells to ADR, VC, or BAF for 1-2 days.Although we saw no DMS after this treatment, it remains pos-sible that extended pulsing of certain chemotherapeutic drugs,by alternatively blocking and releasing DNA replication, con-tributes to the gene amplification necessary for the formationof both abnormal structures (36). Again, the drugs might indi-rectly increase gene amplification by lysing cells and frag-menting chromosomes (37). These fragments may be taken upby cell-contact mediated pinocytosis and amplified by extra-chromosomal replication in drug-selected cells (38), appearingas DMS. Alternatively, DMS may themselves act as analogousintercellular infectious vectors, catalyzing the spread of intrins-ically rare gene amplifications causing drug resistance. Thiswould explain the inexplicably frequent and rapid emergenceof unstable drug resistance encountered in clinical and experi-mental chemotherapy, especially of solid tumors. It is possiblethat the suppression of the propagation, cellular uptake, orchromosomal integration of these new particles would greatlyreduce the incidence and stability of drug resistance, makingpresently marginally effective drug protocols more successfulin destroying tumors.

This work was supported by funds from National Cancer InstituteContract CM 53767, the Leland Fikes Foundation, Inc., and NationalInstitutes of Health Grant CA 21820.

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