[CANCER RESEARCH 26 Part 1, 1397-1407, July 1966]

Amethopterin Resistance in Gloria! Lines of L1210 Mouse Leukemia:Some Associated Biologic and Biochemical Alterations1

AKIRA HOSHINO,2 ALBERTA M. ALBRECHT, JUNE L BIEDLER3, AND DORRIS J. HUTCHISON

Division of Experimental Chemotherapy, Sloan -Kettering Institute for Cancer Research, and Sloan -Ketiering Division, Graduate School ofMedical Sciences, Cornell University Medical College, New York, New York

Summary

The early stages of the development of amethopterin (A)4

resistance in 6 sublines derived from 2 karyotypically differentcell types have been studied. In vivo resistance of greater than16-fold was observed after 3-4 transfer generations. With only 1resistant subline, L26/A3, was there a correlation betweenmaximum in vivo resistance and maximum dihydrofolate reducíaseactivity. In the other sublines 1-5 additional treated transfergenerations were required for the elevation of the reducíaseactivity to its maximum level. Subline L27/A3 showed maximumresistance at generation 3 with no increase in reducíaseactivity,yet after 2 additional treaied iransfer generations ihere was a9-fold increase in enzyme activity. After 20-28 transfers in theabsence of amethopterin the dihydrofolale reducíase activityof L27/A, and L26/A, gradually decreased to lhat of L27 andL26, respectively. However, both sublines remained resistant toamethopterin.

Amethopterin treatment caused chromosomal alterations insome proportions of the cells in all 6 resistant sublines. Thequantitative differences in responses of the amethopterin-resistant sublines to a spectrum of chemotherapeutic drugsindicated that each has its own unique characíerisíicswhichresuli from inherenl differences in íheamethopterin-resistanícells of íhepopulation.

Introduction

A major obstacle in the therapy of leukemia is the emergenceof drug-resistant cells. Several studies have been made recentlyon the mode of action and mechanisms of resistance io amethop-

1This work was supported in part by Grants CA 03192-08 and

5T4 CA 5015 from the National Cancer Institute, NIH, Bethesda,Maryland, and by Grant T-107 from the American Cancer Society.

2Present address: Aichi Cancer Center, Nagoya, Japan.3 USPHS Research Career Development Awardee Grant 1-K3-

CA-5275-02.4The abbreviations used are: A, amethopterin; 6-MP, 6-mer-

captopurine; 5-FU, 5-flourouracil; VLB, vinblastine sulfate;MGGH, methylglyoxal-bis-guanylhydrazone; HU, hydroxyurea;FTJDR, 5-fluorodeoxyuridine; AST, average survival time; SM,submetacentric chromosome; ST, subtelocentric chromosome;1m, minute chromosome; m, small minute chromosome; M,metacentric chromosome.

Received for publication August 9, 1965; revised December 2,1965.

terin (methotrexate) (2, 7, 8, 11, 16, 17, 20, 26, 30). The elevationof dihydrofolate reducíaseactivity as a mechanism of resistancehas been associated with numerous amethopierin-resisiantbiological systems (16, 17). In human leukemia elevated enzymeactivity after amethopterin therapy has been reported (3, 24, 25).

Friedkin et al. (9) described an association between the development of resistance and an increase in dihydrofolate reducíase.Incertain long established lines of LI 210 mouse leukemia wereported (5, 28) an association between the absence of a markerchromosome and an elevated dihydrofolate reducta,ce activity.With other amethopterin-resistant lines we (4) did noi observethe same "all or none" effeci, bui rather noticed populations

with specific alterations of the marker chromosome in 10 out of18 sublines. This paper presents data on the dihydrofolatereducíaseactivity, level of resistance to amethopterin, karyologicstatus, and responsiveness to 6 other chemotherapeutic drugs by6 new sublines of L1210 mouse leukemia during the developmentof resistance to amethopterin with emphasis on the 1st stages ofresistance. The stability of these characteristics after prolongedmaintenance in the absence of amethopterin has been determined.

Materials and Methods

The L1210 (V) mouse leukemia was maintained in DBA2and BDF, (C57BL9 x DBA2c?) mice in the asciiic form asdescribed previously (18). The other amethopterin-sensitivesublines (L26 and L27) used in these experiments were obtainedduring the course of another study in which experiments weredesigned to determine the genetic transformation to amethop-terin, 6-MP, and 5-FU resistance in L1210 cells. Deoxyribonueleicacid was isolated from L1210/A/MP/FU (XVI2) (28) andL1210 (V) cells by the method of Kay et al. (21). Leukemic cellssuspended in tissue culture medium 199 containing spermine (2Mg/ml) and 10% calf serum were incubated in vitro at 37°Cfor 1hr in the presence of the DNA preparations, after which IO6DNA-"treated" cells were implanted i.p. into BDFi mice.

Twenty-four hr afler each in vitro exposure of the L1210 (V)cells to the DXA preparation, groups of 10 mice were challengedwith amethopterin, 6-MP, and 5-FU. After the 14th exposureof cells to each DNA, tumors developed ai íhesite of inoculationand on the greater omentum in mice implanted with the L1210(V) line which had been tested with homologous DNA. Mice notso treated developed ascites but no tumor. The lymphoma wasmaintained for 7 transplant generations as a solid tumor andthen converted to an ascitic form by implanting IO6 cells i.p.into BDFi mice (Chart 1).

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A. Hoshino, A. M. Albrecht, J. L. Biedler, and D. J. Hutchison

LI2IO (V) _]¿WXI (VI INCUBATION

f\ SOLID TUMOR

UNTREATED ASCITES

AMETHOPTERINTREATED ASCITES

L 26 CLONES L 27 CLONE

L26/A

L26/A2

12] L27/A3

L27/A,

TABLE 1

RESPONSE OF L1210 (V) MOUSE LEUKEMIA TOCHEMOTHEKAPEUTICDRUGS"

CHART1. Derivation of amethopterin-sensitive and -resistantL1210 sublines.

CLONALSUBLINES.Karyotype analysis (4, 5) revealed that50% of the population was altered, i.e., possessed a submeta-centric (SM) chromosome rather than the characteristic sub-telocentric (ST) of L1210 (V) and an additional minute chromosome (1m) somewhat larger than the small minute chromosome(m) of the parental line. Several clonal lines were establishedfrom small inocula following the method described recently(4). Two distinctly different cell types, designated L26 and L27,were chosen for study and maintained both in BDF] mice andin a frozen tumor bank according to the method of Hauschkaet al. (13).

AMETHOPTERIN-RESISTANTSUBLINES.Sublines of each clonalline were selected and maintained in vivoaccording to the methodsof Burchenal et al. (6) and Hutchison et al. (18). Three resistantlines were selected by exposure to amethopterin, from the L26population, at transfer generations 9 (L26/A3), 10 (L26/A2),and 12 (L26/AO and 3 lines from the L27 population at transfergenerations 12 (L27/AO, 15 (L27/A8), and 16 (L27/A2).

After transfer generations 34, L26/A! and L27/A] were maintained in vivo in both amethopterin-treated and untreatedanimals. After 20 transfer generations in untreated mice a branchof L27/Ai was maintained in animals treated with folie acid(12 mg/kg).

CHARACTERIZATIONOF THE LEUKEMiAs.Chemotherapy: Theantileukemic effects of amethopterin, 6-MP, 5-FU, VLB, MGGH,HU, and FUDR were determined according to our standardmethods (18). Ten mice were used to assess the activity of each

TRANSFERGEXERATIO.V

No.331

a-5332.^533G._

5DBDG6AAAAAAAFUDRMGGHHUA6-MP5-FUVLB(mg/kg)0.751.53.06.09.012.03.050.0100.0150.03.040.025.01.5Av.

WT.CHANGEC(treated/control)+3.S/-2.7+S.2/-2.7+

1.G/-2.7+1.1/-2.7-1.0/-2.7-0.

8/-2.7+

1.7/+1.2+2.1/+1.2+4.3/+1.2+

1.8/+1.2+0.

S/+2.6-1.S/+2.6-0.S/+2.6+0.4/+2.0AV.

SURVIVALTIME(AST)(treated/

control)10.9/8.811.7/8.814.0/8.819.5/8.822.0/8.820.6/8.815.9/8.79.9/8.714.3/8.710.9/8.714.6/9.414.5/9.420.4/9.416.0/9.4%

CHANGEINAST24335912215013483146425555411770

0 The abbreviations used are: A, amethopterin; 6-MP, 6-mercaptopurine; 5-FU, 5-fluorouracil; VLB, vinblastine sulfate;MGGH, methylglyoxal-bis-guanylhydrazone; HU, hydroxyurea;FUDR, 5-fluorodeoxyuridine.

bTherapy was initiated 24 hr after i.p. inoculation of IO6leu-kemic cells and continued at 48-hr intervals for 10 injections oruntil the animals succumbed. VLB was administered s.c.; allother drugs, by the i.p. route.

" Animals were weighed on the 3rd and 8th days after inoculation; data are presented for the 8th day.

dose for all chemotherapeutic agents. VLB was administereds.c. but all other compounds were injected i.p. The doses areincluded in the appropriate tables and charts. Amethopterinover a wide range of dosages was tested against all lines, including L1210 (V), so that some evaluation of the levels of resistancecould be made on the early transplant generations of the newresistant sublines. A leukemia is considered resistant to a drug ifthere is less than 25 % increase in the average survival time (AST)of treated animals.

KAEYOTYPEANALYSES.Metaphase chromosomal preparationswere made on the 6th day after transplantation. Fifty cells wereexamined and scored as described by Biedler et al. (4, 5). Thelarge bi-armed marker chromosome of the LI 210 cell was classified according to observed differences in length of the shorterarm.

CELL-FREE EXTRACTS AND DIHYDROFOLATE REDUCÕASE ASSAYS.

The procedures used for preparation of extracts of leukemiccells 7 days after transplantation and the quantitative assay fordihydrofoíate reducíase have been described (4). Activity isexpressed in terms of mamóles of dihydrofolate reduced per mgof protein per hr.

Results

The derivation from L1210 (V) and history of the clonal L26and L27 lines and their respective amethopterin-resistant linesare outlined in Chart 1.

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Amethopterin Resistance in L12W Mouse Leukemia

TABLE 2RESPONSETO CHEMOTHEKAPECTICDRUGSDURINGTHE

DEVELOPMENTOF RESISTANCETO AMETHOPTERININ SUBLINEL26/A3"

TABLE 2—CONTINUED

AMETHOPTERIN-TREATED

TRANSFERGENERATION1

(9)>234DRUG

(mg/kg)A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU

150.0Av.

SURVIVALTIME(AST)(treated/control)10.9/7.213.9/7.216.7/7.220.1/7.222.0/7.216.6/7.216.6/7.220.1/7.231.8/7.27.6/7.213.0/7.28.7/7.211.3/9.013.0/9.015.6/9.019.3/9.018.7/9.020.8/9.020.7/9.028.4/9.017.2/9.011.3/9.015.2/9.012.5/9.010.6/9.412.4/9.412.8/9.412.9/9.414.2/9.414.5/9.417.3/9.420.9/9.419.4/9.411.9/9.416.4/9.412.5/9.49.4/9.39.2/9.39.6/9.310.0/9.310.4/9.310.6/9.320.0/9.319.1/9.323.6/9.311.9/9.314.9/9.311.5/9.3%CHANGEIN

AST5193132179206131>131

[3]°179>342

[5]68121264473114108131>130

[1]2169126693913313536505384122>

106[1]2675331-1381214115105154286024

AHETHOPTERIN-TREATED

TRANSFERGENERATION23DRUGAAAAAA6-MP5-FUVLBFUDRMGGHHU(mg/kg)0.751.53.06.0!).()12.040.025.01.550.0100.0150.0Av.

SURVIVALTIME(AST)(treated/control)10.1/10.310.1/10.39.6/10.39.9/10.39.0/10.39.5/10.313.5/10.321.2/10.319.0/10.312.6/10.312.8/10.311.7/10.3%

CHANCEINAST-2-2-7-4-13-831105>85

[1]222414

" The abbreviations are defined in the 1st footnote of Table 1.'Untreated generation from which resistant line (L26/Aj) was

selected.' Numbers in brackets represent 60-day survivors.

L1210 (V). The chemotherapy results with the parental L1210(V) leukemia (Table 1) show an amethopterin dose-responsecurve. Such titration data provided a means of quantitating theamethopterin resistance levels over a 16-fold range. The otherdrugs at the doses used were less effective, which agrees withseveral reports (1, 18, 19, 29).

The dihydrofolate reducíaseactivity (mamóles dihydrofolatereduced/mg of protein/hr) of L1210 (V) at 5 different transfergenerations was 89, 100, 131, 121, and 91 which is in accord withour earlier data (28) and concurrent experiments reported elsewhere (4). The modal number of chromosomes was 40 and all cellsexamined contained 1 ST and l m chromosome as previouslydescribed (5).

CLONALSUBLINEL26. This amethopterin-sensitive subline hada modal chromosome number of 41. There were 2 distinctivemarker chromosomes (Fig. 1), an SM and a 1m (larger than them regularly present in the parental line). This karyotype persisted through 40 transfer generations of the in vivo L26 line.

At transfer generation 7 of L26 (Chart 1) cells were placed inthe tumor bank (-78°C).

In general, the L26 subline was more sensitive than L1210(V) to the spectrum of drugs with the exceptions of FUDR andHU. Chemotherapy data for transplant generation 9 are included in Table 2.

AMETHOPTEKIN-RESISTANT SUBLJNES OF L26. Resistance ¡D

L26/Ai was established at the 4th transfer generation at whichtime the dihydrofolate reducíaseactivity was elevated 3-fold.After an additional 3-fold increase in reducíase activity atgeneration 7, the level remained essentially the same through 31transfer generations (Chart 2).

At transfer generation 5 subline L26/A! was karyotypicallychanged consisting of cells containing either ST, SM, or meta-centric (M) chromosomes (Chart 2). Chromosomal heterogeneitypersisted. At generation 31 most of the cells still contained STchromosomes but there was a higher proportion of cells with Mthan with SM chromosomes.

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A. Hoshino, A. M. Albrecht, J. L. Bicdler, and D. J. Hutchison

=sr

L26/A, D ALTEREDSM

H =SHORT TOP CLASSO

D =ALTERED M

o^> ^DIHYDROFOLATE

REDUCTASE

0 4 8 12 16 20 24 28TRANSFER GENERATION

D=MGGH x=5-FU

32

CHART2. Development of resistance to amethopterin in sublineL26/A]. Lower graph, response to A, amethopterin, 3 mg/kg; HIT,hydroxyurea, 150 mg/kg; FUDR, 5-fluorodeoxyuridine, 50 mg/kg;MGGH, methylglyoxal-bis-guanylhydrazone, 100 mg/kg; VLB,vinbliistinc sulfate, 1.5 mg/kg; 0-MP, O-mercaptopurine, 40mg/kg; and 5-FU, 5-fluorouracil, 25 mg/kg. Upper graph, specificactivity of dihydrofolate reducíase, in mpmnles of dihydrofolatereduced/mg of protein/hr, and distributions of karyotypes duringtreatment with amethopterin.

L26 was sensitive to the spectrum of drugs. L26/Ai, whileretaining sensitivity to 6-MP, 5-FU, and VLB, had becomecross-resistant to MGGH by transfer generation 23. At generation30 similar results were obtained. HU was slightly effective againstL26 while the subline L26/Ai was resistant (Chart 2).

L26/A2. After L26 cells were removed from the bank andpassed for 2 generations in untreated mice, selection of L26/A2by amethopterin was started (Chart 1). A small percentage ofthe population consisted of ST-containing cells. The modalnumber was 41 and the additional 1m chromosome was present.The chromosomal pattern of generation 12 was markedly altered:an M chromosome (Fig. 2) was present, the SM had disappearedand more than 0.5 of the population contained the ST chromosome (Chart 3).

L26/A2 was resistant to amethopterin with a greater than 16-fold increase at transfer generation 4 when simultaneously thedihydrofolate reducíaseactivity was elevated 14-fold (Chart 3).Whereas the line became progressively less sensitive, though notcross-resistant, to 6-MP and VLB (Chart 3), it retained itsoriginal responsiveness to 5-FU, MGGH, and HU and exhibitedsome collateral sensitivity to FUDR.

L26/A3. This amethopterin-resistant subline (Chart 1) wasalso obtained from a population of cells that had been stored in

the tumor bank for several weeks. The population of L26 (generation 9) was heterogeneous; i.e., 86% of the cells contained an SMchromosome and 14% an ST chromosome. The cells had a modalnumber of 41 and contained the 1m chromosome. The karyotypicpattern of L26/A3 fluctuated with a pronounced shift towardST-containing cells (Chart 4). Table 2 shows that resistance toamethopterin in subline L26/A3 increased 2-fold at generation 2,16-fold at generation 3, and greater than 16-fold at generation 4.

The dihydrofolate reducíase activity of L26 (generation 9) was47 or about 0.5 the value obtained with the other population ofL26 cells. However, the relative increase at generations 2, 3,and 4 of subline L26/A3 was 3-, 4-, and 8-fold, respectively(Chart 4).

L26/A3 was marginally sensitive to FUDR and HU (Table 2).It remained sensitive to 5-FU but became progressively less soto 6-MP and VLB. It was cross-resistant to MGGH by genera

tion 23.CLONAL SUBL1XE L27. This amethopterin-sensitive subline

(Fig. 3) had a modal chromosome number of 40 with a karyotypeapparently identical to L1210 (V) (5). At generation 13 somecells were frozen and stored. The L27 subline was generally moresusceptible to the therapeutic drugs than L1210 (V). Details ofthe response to drugs of transplant generation 15 are shown inTable 3. FUDR and HU were ineffective.

•=ST H=SHORT TOP CLASS 0H=i-26SM O-ALT ERE D M

UNALTERED SM o-o=D/HrDROFOLATE

L26/A2 REDUCTASE

100

75-

%

O 4 8 12 16 20TRANSFER GENERATION

CHABT3. Development of resistance to amethopterin in sublineL26/A2 (see legend for Chart 2).

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Amethopterin Resistance in L1210 Mouse Leukemia

•ST-- L26 SM

=ALTERED SM

M =SHORT TOP CLASS 0

CH =ALTERED M

°-°=DIHYDROFOLATE

REDUCTASE

2000XK

1500 §

]000

500

o

oCoklO.to

I lO 2 4 6 8 IO 12 14 16 18 20 22 24 26

TRANSFER GENERATION

CHART4. Dihydrofolate reducíaseactivity and karyotype distribution during development of amethopterin resistance in sublineL26/A3.

AMETHOPTERIN-RESISTANT SUBL1NES OF L27. L27/\i became

resistant at treated transfer generation 3 at which time thekaryotype of the population was unaltered (Chart 5). However,during the 5th-8th transfer generations pronounced heterogeneity

was observed. At generation 9 the population returned to theoriginal karyotype and remained homogeneous.

The dihydrofolate reducíaseactivity exhibited a 3-fold increaseat generation 4, and a further 3-fold increase at generation 8. Itremained at this level throughout the experiment (Chart 5).Chemotherapy assays carried out at generations 14, 23, and 30showed that the L27/Ai line retained sensitivity to 6-MP,5-FU, and VLB and became cross-resistant to MGGH (Chart 5).

L27/A2. The L27 cells that had been frozen at transfer generation 13 for several weeks were passed for 2 generations in untreated mice. Then the L27/A2 selection was started (Chart 1).The karyotypic picture of generation 16 was identical with thatof the in vivoline (Chart 6). However, at the 2nd treated transfergeneration of L27/A2 a new and distinctive short armed markerchromosome (Fig. 4) was observed. This chromosome is identified as belonging to class 0 (4), where the short arm is conspicuously shorter than that of the ST marker. At treatedgeneration 12 about 0.5 of the cells contained this altered chromosome (Chart 6).

Resistance to amethopterin increased 2-fold at transfergeneration 2, at least 4-fold at generation 3, and greater than16-fold in transfer generation 4 (Chart 6). The dihydrofolatereductase activity was elevated 14-fold at generation 4, at whichlevel it remained (Chart 6).

There was little change in sensitivity to drugs, other than toVLB. Marked collateral sensitivity to VLB was seen at transfergenerations 6, 8, and 9. However, at transfer generation 11,L27/A2 had essentially the same sensitivity to VLB as did L27.

L27/A3. This subline (Chart 1) had a karyotypically homogeneous population which became altered at the 3rd treatmentgeneration. M-chromosome-containing cells (Fig. 5) appeared

and persisted in various proportions (Chart 7). At transfergeneration 2 there was a 2-fold increase in resistance to amethopterin and a greater than 16-fold increase at generation 3 (Table3). Dihydrofolate reductase activity did not increase untiltransfer generation 4 when there was a 6-fold elevation, followedby a 9-fold elevation at generation 5 (Chart 7).

L27/A3 remained susceptible to 5-FU throughout and lost somesensitivity to 6-MP, VLB, and MGGH. This subline showedslight but definite increase in response to FUDR (Table 3).

STABILITYOF RESISTANCE.After 34 treated transfer generations of L26/Ai and L27/Ai each line was also maintained inuntreated mice for 28 or more additional generations. L27/Ajwas also carried in folie acid-treated animals for 50 transfergenerations. Dihydrofolate reducíasegradually decreased (Table4). The untreated subline L26/Ai at generation 28 had the sameactivity as the L26 population. The L27/Ai untreated sublineand the folie acid-treated subline also lost enzyme activity. Thelevels of reductase activity were the same as for the originalL27 population at the 20th transfer generation. Both untreatedsublines (Table 5) were still completely resistant to amethopterinand retained their same sensitivity to the other drugs. Essentiallyidentical data were obtained with the folie acid-treated subline.No karyotypic change was observed in the untreated L27/Aisubline. However, the population of the L26/Ai untreatedsubline at generation 29 was altered; 36% of the cells containedthe SM chromosome but the additional 1m chromosome wasabsent (Fig. 6).

Table 6 shows that when the untreated line L27/Ai (not treatedfor 24-26 generations) was treated with amethopterin, within 3generations the dihydrofolate reductase activity was equivalentto the high level of the treated L27/Ai line at generation 8(Chart 5). The rate of increase varied little among the 3-doseschedules of amethopterin.

Folie acid (12 mg/kg X 3) for 3 transfer generations (Table6) did not induce increased dihydrofolate reductase activity.

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A. Hoshino, A. M. Albrecht, J. L. Ricdlcr, and D. J. Hutchison

TABLE 3

RESPONSE TO CHEMOTHEKAPEUTICDRUGS DURING THEDEVELOPMENTOF RESISTANCE TO AMETHOPTERIN

IN SUBLINE L27/A3°

TABLE 3—CONTINUED

AMETHOPTEBIN-TREATED

TRANSFERGENERATION1

(15)'234DRUG

(mg/kg)A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0A6.0A9.0A12.0G-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0AG.OA9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU150.0A

0.75A1.5A3.0A6.0A9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU

150.0AV.

SURVIVALTIME(AST)(treated/control)9.8/7.612.8/7.616.0/7.623.3/7.624.4/7.617.2/7.622.3/7.613.0/7.612.1/7.68.1/7.613.1/7.69.2/7.612.7/9.314.3/9.3Ki.7/9.317.0/9.315.8/9.312.6/9.311.1/9.313.1/9.310.6/9.312.2/9.311.7/9.310.4/9.310.5/9.211.0/9.211.3/9.211.7/9.211.8/9.210.1/9.213.3/9.215.1/9.212.6/9.211.6/9.212.6/9.211.2/9.29.0/8.49.1/8.49.1/8.49.7/8.49.0/8.49.6/8.411.8/8.413.6/8.414.3/8.412.3/8.49.4/8.410.4/8.4%CHANGEIN

AST2968111207221126>193

[1]«71597722137548083703619411431271214192327271047643626362278816714416270461224

AMETHOPTERIN-TREATED

TRANSFERGENERATION23DRUG

(mg/kg)A

0.75A1.5A3.0A(i.OA9.0A12.06-MP

40.05-FU25.0VLB1.5FUDR50.0MGGH100.0HU

150.0AV.

SURVIVALTIME(AST)(treated/control)9.1/9.69.2/9.69.9/9.69.2/9.69.6/9.68.4/9.613.1/9.616.5/9.612.9/9.613.1/9.612.5/9.610.8/9.6%CHANGEIN

AST-5-43-40-13377234373013

" The abbreviations are defined in the 1st footnote to Table 1.0 Untreated generation from which resistant line (L27/A3) was

selected." Numbers in brackets represent 60-day survivors.

•*STH=L26

OALJERED SM

a=SHORT TOP CLASS 0G= ALTERED M

2000 >

1500 o<i1000 o

500 Gui

O fi.

-20324 8 12 16 20 24 28

TRANSFER GENERATION•=A D,MGGH x=5-FUo =HU A=W_8 A=6-MP•=FUdR

CHARTÖ.Development of resistance to amethopterin in sublineL27/Ai (see legend for Chart 2).

During 4 additional folie acid-treated generations there was no

increase in reducíase activity; in fact, there was no increasefollowing single injections of amethopterin (12 mg/kg), folie acid(12 mg/kg), or folinie acid (24 mg/kg).

Discussion

The numerous aspects of antifolate resistance and the implicated possible and impossible mechanisms whereby some 50different cell populations express their lack of responsiveness toamethopterin have been presented in some detail in 2 reviews

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Amethopterin Resistance in L1210 Mouse Leukemia

L27/A2lOOr

= ST B =SHORT TOP CLASS 0=L26SM H--ALTEREDM=ALTERED SM o-o=DIHYDROFOLATE

REDUCTASE

O 4 8 12 16 20TRANSFER GENERATION

a=MGGHÛ--6-MP

CHAKT 6. Development of resistance to amethropterin in sub-line L27/A2 (see legend for Chart 2).

(16, 17). The multiplicity of the ways in which this phenomenonpresents itself is indeed a riddle. The solution of this cannot beobtained until the nature of the system from which the resistantpopulation arises is completely understood. The emergence of aresistant population is dependent on factors such as numbers ofcells, sensitivity of these cells, frequency of exposure to the drug,concentration of the drug, length of exposure to the drug, andmutagenicity of the drug. The normal mutation rate and homogeneity of populations also have considerable influence on thedevelopment of resistance. The studies presented here weredesigned to examine certain aspects of these factors.

Certainly the rate at which resistance to amethopterin evolvesis a major problem.

Attendant with resistance to amethopterin is the matter ofcross-resistance to other useful drugs (16, 17). However, if aconcomitant increase in sensitivity to other drugs or evenretention of sensitivity to other drugs is associated witli resistancethe problem may not be so serious. Furthermore, if such biologicalterations could be coupled with specific cytogenetic modifications, the general responsiveness of the population could bepredicted. To determine if there were any association of dihydro-folate reducíaseactivity with these additional aspects of biologicactivity, 2 clonal lines of different karyotypic make-up (Figs. 1,3) were chosen. To obtain a more specific concept of the development of resistance to amethopterin, 3 resistant sublines wereselected from each of the donai lines as near the same in vivotransfer generation as was feasible. For this purpose certaininocula were stored in the frozen tumor bank. Some changes mayhave resulted from freezing and storage. For example, the L26population was apparently homogeneous (SM) at the time offreezing, but when 2 different samples were removed from thetumor bank, the derived populations consisted of both SM- andST-containing cells. This suggests that the original L26 inoculumwas not a single cell and furthermore that the SM cells were lessable to withstand freezing than the ST-containing cells.

The 3 amethopterin-resistant sublines derived from 3 different

r~l = L26 SM

CD =ALTERED SM

% 50

0 2

• =SHORT TOP CLASS 0

II =ALTERED M

DIHYDROFOLATEREDUCTASE

2000XK

1500 >I-O•Õ

1000o

500 gCLen

o

468 10 12 14 16 18 20 22 24 26

TRANSFER GENERATION

CHART 7. Dihydrofolate reducíase activity and karyotype distribution during development of amethopterin resistance in subline

L27/A3.

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A. Hoshino, A. M. Albrecht, J. L. Biedler, and D. J. Hutchison

TABLE 4DECREASEIN DIHYDROFOLATEREDUCTASEACTIVITYm L26/A]

ANDL27/Ai POPULATIONSIN THE ABSENCEOFAMETHOPTERIN

TABLE 5RESPONSEOF SUBLINESL26/Ai ANDL27/Ai, AFTERMAINTENANCEIN THE ABSENCEOF AMETHOPTERIN,

TO CHEMOTHERAPEUTICDRUGS»

NO. OF GENERATIONSWITHOUT

AMETHOPTERINTREATMENT135101214181!)2024284150DIHYDROFOLATE

REDUCTASEACTIVITY*1L26/A,1489102910(1058458633519392L27/A,848109254156826525312915911794112111L27/Ai6924136120101

" m/¿niolcsof dihydrofolate reduced/ing of protein/hr.6Treated with folie acid, 12 mg/kg.

L26 transfer generations were resistant at the 3rd or 4th transfergenerations. The more quantitative data on L26/A3 (Table 2)show a general increase in in vivo resistance with a concomitantincrease in reducíase activity (Chart 4) and the maximumelevation of reducíasecoincided with maximum in vivoresistance.In the other 2 sublines reductase activity increased after completein vivo resistance had occurred (Charts 2, 3).

The 3 sublines selected by amethopterin from the L27 population were totally resistant at transfer generation 3 (Charts 5, 6)(Table 3). In the L27/A3 subline there was no concomitantincrease in dihydrofolate reductase. However, after 1 additionaltransfer generation enzyme activity increased 6-fold and, after 2generations, 9-fold (Chart 7).

In 1 instance (L26/A3) maximum reductace activity corresponded with maximum in vivo resistance, which is in accordwith the results (9, 10) of Friedkin's group, while in the other

(L27/A3) the resistance in vivo was apparent 2 transfer generations before maximum reductase activity.

Cross-resistance to MGGH occurred in L26/Ai, L26/A3, andL27/Ai. Responses to the other drugs (6-MP, 5-FU, FUDR, HU,and VLB) for the 6 sublines were essentially unchanged afteramethopterin resistance was established, except for L27/A2 inwhich marked collateral sensitivity to VLB was seen at generations 6, 8, and 9. The short armed ST chromosome (Fig. 4)appeared at generation 2. At generation 11, the response ofL27/A2 to VLB reverted to that of the sensitive population, at atime when more than half of the cells were karyotypicallyaltered (Chart 6).

It is significant and encouraging that cross-resistance to 6-MP,5-FU, and VLB does not occur early in the amethopterin-treatedpopulation. Indeed, in every line there were varying rates ofdecrease in response to the other agents.

In the 6 amethopterin-resistant sublines chromosomal changesoccurred in some proportions of the cells, and ST-containingcells were present in the majority of the populations. Although

SUBLIKE. NO. OFUNTREATED

GENERATIONSL26/A,,

28L27/A,,

23L27/A,,

28DRUG

(mg/kg)AAAAAA6-MP5-FUVLBFUDRMGGHHUAAAAAAA6-MP5-FUVLBFUDRMGGHHU0.751.53.06.09.012.040.025.01.550.0100.0150.00.751.53.06.09.012.03.040.025.01.550.0100.0150.0Av.WT.CHANGE(treated/control)-0.9/-0.5+0.5/-0.5+0.8/-0.5-0.8/-0.5-2.4/-0.5-3.3/-0.5-1.2/-0.50/-0.5+0.3/-0.5-2.1/-0.50/-0.5-3.4/-0.5-0.8/-1.5+

1.2/-1.5+2.2/-1.5+

1.1/-1.5+0.1/-1.5-3.0/-1.5+2.2/-0.9-1.9/-0.9-1.6/-0.9-0.8/-0.9-0.9/-0.9-0.7/-0.9-2.7/-0.9Av.

SURVIVALTIME(AST)(treated/

control)10.5/9.510.0/9.510.7/9.510.3/9.510.6/9.59.9/9.515.2/9.516.7/9.517.5/9.513.0/9.512.2/9.511.0/9.59.4/10.611.8/10.611.4/10.611.6/10.611.7/10.69.9/10.610.9/9.614.2/9.616.1/9.612.7/9.611.7/9.612.2/9.611.3/9.6%CHANGEINAST1151381246076843728169118910-714486832222718

" The abbreviations are defined in the 1st footnote to Table 1.

TABLE 6THE EFFECTOF AMETHOPTEHIN(A) ANDFOLICACIDTREATMENT

ONTHEDIHYDROFOLATEREDTCTASEACTIVITYOFTHESUBLINEL27/Ai MAINTAINEDIN THEABSENCEOFAMETHOPTERIN

UNTREATEDGENERATIO.VOFL27/A24242620DRCG

(mg/kg)A

0.75A1.5A3.0Folie

acid 12.0DAY

OFTREATMENT1,

2, 3, 4,51,2, 3, 4,51,3,51,3, 5DIHYDROFOLATE

REDUCTASEACTIVITY11No.

of transfergenerationstreated09494117i370239109922702588219116397398678799

0 Dihydrofolate reduced (m/«moles)/mgof protein/hr.

this is not in agreement with our results on the long maintainedline L1210/A/MP/FU XVI2 (5) certain correlations can be madewith chromosomal alterations in 2 other resistant lines (4). Thepopulations of Q-10 (4) and L27/Ai (Chart 5) originated asST-containing cells, but after several transfer generations duringwhich karyotypic heterogeneity was marked, the cells reverted to

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Amethopterin Resistance in L1210 Mouse Leukemia

ST populations. In each line the in vivoresistance was similar andredactase activity was elevated although quantitatively different.The karyotypes of both L26/A2 (Chart 3) and Q-17 (4) populations after 12 or more transfer generations were essentiallyidentical. Again, both lines were equally resistant to amethopterinbut exhibited a quantitative difference in reducíaseactivity.

Friedkin et al. (9) and Schrecker and Greenberg (27) examinedthe stability of resistance to amethopterin during the absence ofamethopterin treatment as measured both by in vivotherapy andreducíaselevels. They found dihydrofolate reducíaseactivitydecreased gradually to the level of ihe sensitive, parental population with no indication of in vivosensitivity. The resulls wiih theuntreated L26/A! and 127/\¡ lines (Table 5) agree with thesestudies. In addition the response of both lines to other drugswas unaltered even after 28 untreated generations. Whereas thekaryotypic picture of untreated L27/Ai was constant, thai ofuntreated L26/Ai changed. This change may have arisen withinthe ST cells themselves, contained in the L26 population sincethe additional 1m chromosome characterizing the SM cells ofL26 was not present. Although the possibility of ihe presence ofthis karyolype (Fig. 6) in the original inoculum cannot beeliminated, it appears likely that the alteration may have been aresult of exposure of ST cells to amethopterin.

With the L27/Ai untreated line, attempts were made to inducedihydrofolate reducíaseformation by folie acid and folinic acidbut no increase in activity occurred after treatments with either.However, treatment with amethopierin for 3 generations (Table6) caused increased reducíaseequivalent to that of the L27/Aisubline (Chart 5) in which the maximum reducíaseactivity didnot appear until treated generation 8. This suggests ihal iheuntreated population of L27/A] was helerogeneous in respect tosensitive and resistant cells even though the cells appeared ihesame karyotypically. The disappearance of resislani cells, withdihydrofolate reducíaseactivity as the indicator of resistance, inthe absence of treatment, from mixed populations of sensitiveand resistant cells has been observed (14). The rapid increase inreducíasefollowing ameihopterin treatment also suggests IhalIhe population was mixed in respect to cells with high and lowenzyme activity. One resistant cell per million sensitive cells willresult in a resistant population in 2 generations of treatmentwhile the reducíasewill be increased only 3-fold (15).

Berlino et al. (3) showed Ihal Ihe dihydrofolate reduélaseofhuman leukemic cells was inducible by amethopterin. Hakalaet al. (12) also presented evidence for a rapid increase in folatereducíasein S-180 cells afler exposure to ameihopterin. Possiblyin those systems Ihe continued presence of the inducer was notnecessary to maintain high enzyme activity. Our data indicatethat a selection by ameihopterin based on mutation rate ofbelween 10~6and 10~7 is Ihe explanation for the resistance and

the increased enzyme activity. The fact that neilher folie acidnor folinic acid Irealmenl caused the same increase in enzymeactivity further supports a selection process.

Only 2 pairs of resistanl L1210 lines [L26/A2 and Q-17 (4) andL27/Ai and Q-10 (4)] were identical in sequence, proportions,and types of altered cells, and Ihese similar populations differedin reducíase activity. We can, therefore, speculate that thespecific genetic modification responsible for the resistance toamethopterin is a mutalion, sometimes but not necessarilyinvolving a visible chromosomal change. The results of Klein

(22) on the rapid emergence of resistanl populations from smallinocula of L1210 can also be interpreted as being a result of amutagenic action of amethopierin in conjunction with hisvariation-selection model.

From our data it appears that amethopierin as suggested (4)is a mutagenic agenl and, as such, alters Ihe chromosomes.Lindner et al. (23) reported marked damage to the chromosomesof Ehrlich asciles after exposure lo amethopierin. Ten of the 18amelhopterin-trealed lines of Biedler et al. (4) were altered.Furthermore, in our 6 resislani lines karyotype analysis revealeddefinite heterogeneily, whereas the untreated, parental population of a number of L1210 lines (4), including the 2 parentalpopulations, L26 and L27, which were never frozen were homogeneous and have remained homogeneous. Therefore, the treatmentwith amethopterin was more than likely responsible for thechromosomal changes as well as certain changes in geneticmaterial thai are too small to be seen as structural alterations.The nature of this action of amethopterin should be furtherinvestigated.

Some quantitative aspects of the composition of the cellpopulations in respect lo relative numbers of amelhoplerin-sensitive and -resistanl cells have been determined (15).

Acknowledgments

The excellent technical assistance of Mr. Dennis L. Robinson,Mrs. Jane L. Palmer, and Miss Francesca Marshall is acknowledged.

References

1. Adamson, R. H., Oliverio, V. T., Derham, C., and Venditti,J. M. Concentration of Methyl-bis-guanylhydrassone-C14 in

Mouse Leukemia L1210 and a Resistant Variant. Life Sci.,7: 493-96, 1963.

2. Berlino, J. R. The Mechanism of Action of the Folate Antagonists in Man. Cancer Res., 23: 1286-1306, 1963.

3. Berlino, J. R., Donohue, D. M., Simmons, B., Gabrio, B. W.,Silber, R., and Huennekens, F.M.The 'Induction' of Dihydro-

folic Reducíase Activity in Leukocytes and Erythrocytes ofPatients Treated with Amethopterin. J. Clin. Invest., 42:466-75, 1963.

4. Biedler, J. L., Albrecht, A. M., and Hutchison, D. J. Cyto-genetics of Mouse Leukemia L1210. I. Association of a SpecificChromosome with Dihydrofolate Reducíase Activity inAmethopterin-treated Sublines. Cancer Res., 25: 246-57, 1965.

5. Biedler, J. L., Schrecker, A. W., and Hutchison, D. J. Selectionof Chromosomal Variant in Amethopterin-resistant Sublinesof Leukemia L1210 with Increased Levels of DihydrofolateReducíase. J. Nati. Cancer Inst., 31: 575-601, 1963.

6. Burchenal, J. H., Robinson, E., Johnston, S. F., and Kushida,M. N. The Induction of Resistance to 4-Amino-A"°-Methyl-

Pleroylglutamic Acid in a Strain of Transmitted Mouse Leukemia. Science, ;//: 116-17, 1950.

7. Fischer, G. A. Increased Levels of Folie Acid Reducíase as aMechanism of Resistance to Amethopterin in Leukemic Cells.Biochem. Pharmacol., 7: 75-77, 1961.

8. Friedkin, M. Enzymatic Aspects of Folie Acid. Ann. Rev.Biochem., 32: 185-214, 1963.

9. Friedkin, M., Crawford, E., Humphreys, S. R., and Goldin, A.The Association of Increased Dihydrofolate Reducíase withAmethopterin Resistance in Mouse Leukemia. Cancer Res.,22: 600-6, 1962.

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A. Hoshino, A. M. Albrecht, J. L. Biedler, and D. J. Hutchison

10. Friedkin, M., and (¡oldin,A. The Use of Dihydrofolate Reducíase in Studies of Mixed Populations of Sensitive and Resistant Leukemic Cells. Ibid., 22: 607-16, 1962.

11. Hakala, M. T., and Ishihara, T. Chromosomal Constitutionand Amethopterin Resistance in Cultured Mouse Cells. Ibid.,Si: 987-92, 1962.

12. Hakala, M. T., Zakrzewski, S. F., and Nichol, C. A. Relationof Folie Acid Reducíase to Amethopterin Resistance in Cultured Mammalian Cells. J. Biol. Chem., 236: 952-58, 1961.

13. Hauschka, T. S., Mitchell, J. T., and Niederpruem, D. J. AReliable Frozen Tissue Bank: Viability and Stabilily of 82Neoplaslic and Normal Cell Types Afler Prolonged Storageat -78°C. Cancer Res., 19: 643-53, 1959.

14. Hoshino, A. Albrecht, A. M., Biedler, J. L., and Hutchison,D. J. Disappearance of Resistant Mutants in Leukemic CellPopulations. Proc. Am. Assoc. Cancer Res., 5: 29, 19(54.

15. Hoshino, A., Albrecht, A. M., and Hutchison, D. J. Fate ofAmethopterin-resislant Mutants in L1210 Mouse LeukemiaPopulations. Cancer Res., 26: 974-78, 1966.

16. Hutchison, D. J. Cross-resistance and Collateral SensitivityStudies in Cancer Chemotherapy. Advan. Cancer Res., 7:235-350, 1963.

17. — —. Studies on Cross-resistance and Collateral Sensitivity(1962-1964). Cancer Res., 25: 1581-95, 1965.

18. Hutchison, D. J., Robinson, D. L., Martin, D. Ittensohn, O.L., and Dillenberg, J. Effects of Selected Cancer Chemo-therapeutic Drugs on the Survival Times of Mice with L 1210Leukemia: Relative Responses of Antimetabolite ResislantStrains. Ibid., 22: 57-72, 1962.

19. Johnson, I. S., Armstrong, J. G., Gorman, M., and Burnett,Jr., J. P. The Vinca Alkaloids: A New Class of OncolyticAgents. Ibid., US: 1390-1427, 1963.

20. Jukes, T. H., and Broquist, H. P. Sulfonamides and Folio

Acid Antagonists. In: R. M. Höchster and J. H. Quastel (eds.),Metabolic Inhibitors, Vol. 1, pp. 481-534. New York: Academic

Press, Inc., 1963.21. Kay, E. R. M., Simmons, N. S., and Donnée,A. L. An Im

proved Preparation of Sodium Desoxyribonucleate. J. Am.Chem. Soc., 74: 1724-26, 1952.

22. Klein, G. Variation and Selection in Tumor Cell Populations.Can. Cancer Conf., 3: 215-240, 1959.

23. Lindner, A., Kutkam, T., Cue, D., Corey, B., and Toliver, H.Biological Studies on Sites of Metholrexate (MTX) Action.Proc. Am. Assoc. Cancer Res., 5: 41, 1964.

24. Roberts, D., and Hall, T. C. Folie Reducíase Content ofHuman and Animal Tissues. Ibid., 4-'57, 1903.

25. — •.Folie Reducíase Aclivily of Human While Blood Cells.Ibid., 5:54,1964.

26. Sartorelli, A. C., Booth, B. A., and Berlino, J. 11. FolateMetabolism in Methotrexate-sensilive and -Resistant EhrlichAscites Cells. Arch. Biochem. Biophys., 108: 53-59, 1964.

27. Schrecker, A. W., and Greenberg, N. H. Change of Dihydro-folic Reducíase Levels in Leukemia L1210 During Development and Loss of Antifolate Resistance. Proc. Am. Assoc.Cancer Res., S: 56, 1964.

28. Schrecker, A. W., Venditti, J. M., Greenberg, N. H., Biedler,J. L., Robinson, D. L., and Hutchison, D. J. Association ofIncreased Dihydrofolate Reducíase Levels and ChromosomeAlteralion in Amelhoplerin-resistant Sublines of LeukemiaL1210. J. Nail. Cancer Insl., 31: 557-74, 1963.

29. Slearns, B., Losee, K. A., and Bernstein, J. Hydroxyurea. ANew Type of Potential Antitumor Agent. J. Med. Chem., 6:201, 1963.

30. Werkheiser, W. C. The Biochemical, Cellular, and Pharmacological Aclion and Effecls of ihe Folie Acid Anlagonists. Cancer Res., 23: 1277-85, 1963.

FIG. 1. Metaphase cell of clonal subline L2(i with ihe submelacenlric (SM), minule (m), and additional larger minute (1m) markerchromosomes; 41 chromosomes. X 1600.

FIG. 2. Cell of subline L26/A»with an M marker and both minute chromosomes. X Ki(X).FIG. 3. Representative cell of clonal subline L27 with the subtelocentric (ST) and m marker chromosomes; 40 chromosomes. X 1600.FIG. 4. Cell of L27/Aîwith the dislinclive bi-armed marker (class 0) and ihe regular m chromosome. X HiOO.FIG. 5. Cell of L27/A»with M and m marker chromosomes. X 1600.FIG. 6. Cell of L26/Ai (untreated generation 29) with the SM and m chromosomes, but lacking the additional 1m marker; 40 chromo

somes. X 1600.

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Amethopterin Resistance in L1210 Mouse Leukemia

"A'«À

»*• »** .*. *****

1

JULY 1966 1407

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1966;26:1397-1407. Cancer Res   Akira Hoshino, Alberta M. Albrecht, June L. Biedler, et al.   AlterationsLeukemia: Some Associated Biologic and Biochemical Amethopterin Resistance in Clonal Lines of L1210 Mouse

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