1983 62: 180-185   

 F Carr, WD Medina, S Dube and JR Bertino resistanceGenetic transformation of murine bone marrow cells to methotrexate

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Genetic Transformation of Murine Bone Marrow Cells to

180 Blood, Vol. 62, No. 1 (July), 1983: pp. 180-185

Methotrexate Resistance

By Frank Cam. William D. Medina, Shyam Dube, and Joseph R. Bertino

Genetic transformation of murine bone marrow stem cells

to methotrexate resistance was achieved using a modified

calcium phosphate-DNA coprecipitation procedure. Bone

marrow cells were transformed by DNA derived from

methotrexate-resistant mouse 3T6 cells. In vivo selection

of drug-resistant bone marrow cells resulted from thrice

weekly injections of methotrexate (MTX) for a period of

6-8 wk. Following selection. dihydrofolate reductase activ-

ity encoded by the donor DNA species was easily detect-

able in extracts of recipient mouse spleens. In addition.

A NOVEL IDEA for reducing the toxicity of van-

ous anticancer drugs to bone marrow cells is to

induce drug resistance in these cells. The experiments

of Cline and coworkers’ suggested that methotrexate

(MTX) resistant murine bone marrow cells could be

selected for in vivo following injection of cells treated

in vitro with DNA from a MTX-resistant mouse cell

line (3T6). MTX-resistant bone marrow cells were

routinely observed in different mice in these expeni-

ments, despite the expected low frequencies of trans-

formation to MTX resistance (usually less than lO�

with cell lines in tissue culture).2 The induction of

resistance to MTX in the majority of mice, together

with the presumably small numbers of pluripotent

stem cells injected, suggested that the efficiency of

transformation for these cells was higher than for most

established cell lines. The generality of transformation

of bone marrow cells to MTX resistance has been

further investigated, and an improved transformation

procedure has been developed.

C�ells

MATERIALS AND METHODS

Bone marrow cells were obtained from femurs of 6-wk-old female

CBA and CBA/H-T6T6 mice (Jackson Laboratory, Bar Harbor,

ME) by flushing the marrow cavity with ice-cold McCoy’s SA

medium supplemented with 10% fetal calf serum (FCS, GIBCO,

Grand Island, NY) and I U/mI hepanin. Donor mice were treated

with 4 mg/kg vinblastine 3 days prior to sacrifice to increase the

number of marrow hemopoietic stem cells.3 Chinese hamster ovary

(CHO) wild type and MTX-resistant A29 cells2 (obtained from Dr.

From the Departments of Pharmacology and Medicine. Yale

School ofMedicine, New Haven, CT.

Supported in part by Research Grant CA 28852; J.R.B. is an

American Cancer Society Professor.

Submitted July 27, /982; accepted January 19, /983.

Address reprint requests to Dr. J. R. Bertino, Yale University

School of Medicine, Department of Pharmacology. 333 Cedar

Street, New Haven, CT 06510.

© I 983 by Grune & Stratton, Inc.

0006-497//83/6/06-0026$Ol.00/0

selection of methotrexate-resistant cells was indicated by

the persistence of spleen colony-forming units (CFU-S) in

drug-treated animals. Also. changes in ratios of mixed

syngeneic bone marrow cells derived from CBA and CBA/

T6T6 mice resulted from initial treatment of either cell

type with 3T6 DNA. These results confirm and extend the

observations of Cline and coworkers that normal bone

marrow cells can be genetically transformed to methotrex-

ate resistance.

L. Siminovitch) were grown in Dulbecco’s modification of Eagle’s

medium (DMEM) containing 10% FCS and 3x normal nonessential

amino acids. A29 cells (Pro 3 MTXRIII2) possess an increased

activity of mutant form of CHO dihydrofolate reductase (DHFR)

activity, which is more resistant to MTX inhibition than the wild

type enzyme.4 Mouse embryo 3T6 fibroblast cells (obtained from

Dr. R. Kellems) weregrown in Dulbecco’s MEM with 10% FCS and

4 x l0�� M MTX. These cells contain high levels of mutant DHFR,

which has reduced affinity for MIX compared to wild type enzyme.�

L5178Y cells (single copy, MTX-sensitive DHFR) were grown in

Fischer’s medium containing 10% horse serum. Prior to preparation

of DNA or chromosomes, CHO, 3T6, and L5178Y cells were

propagated as suspensions in spinner flasks.

The cell line, 416B (obtained from Dr. E. Scolnick and Dr. M.

Moore), is a biopotential murine hemopoietic line derived from

cultures (C57BL/6 x DBA/2)F of bone marrow cells that were

infected with Friend leukemia virus.6 The 416B cells were grown in

Fischers medium supplemented with 20% horse serum. Human

lymphoblast CCRF-CEM cells (obtained from H. Lazerus) were

grown in RPMI 1640 medium containing 10’�f horse serum.

DNA and Chromosome Preparations

DNA was prepared by a modification of the method of Pellicer

and coworkers;’ this procedure involves extraction of aqueous cell

lysates three times with phenol and then three times with ether.

Purified DNA preparations were at least 69 Kb in size, as estimated

after electrophoresis in agarose gels. Chromosomes were isolated

from CHO and A29 cells by a modification of the method of Lewis

and coworkers.’ Following disruption of cells by homogenization,

chromosome preparations were layered onto a shelfof I M sucrose in

homogenization buffer and centrifuged at 100 g for 10 mm. The

sucrose-buffer interface layer was then removed and diluted before

applying to another I M sucrose shelf. The chromosomes were then

pelleted at I ,000 g for 20 mm, and finally washed and resuspended in

H EPES-buffered saline prior to transformation.

Genetic Transformation of Cells

Initial treatment ofcells with DNA or chromosome coprecipitates

with calcium phosphate was based on the modification of Wigler and

coworkers.’#{176} For bone marrow cell experiments, 2 x 106 cells/mI in

50 ml McCoy’s 5A medium containing 10% FCS were treated

directly in a TI 50 flask with 5 ml of calcium phosphate coprecipitate

with DNA (100 �.Lg) or chromosomes (1-2 cell equivalents). The

cells were incubated for 4 hr and then pelleted and resuspended in

fresh medium. This suspension also included particles of the micro-

precipitate, and in preliminary experiments, injection of this suspen-

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MTX-RESISTANT MURINE BONE MARROW CELLS 181

sion resulted in the rapid death of recipient mice. Therefore, the

concentrated cell suspensions were layered onto a shelf of 9.6%

(w/v) sodium metnizoate (Lymphoprep, Accurate Scientific and

Chemical Company, New York) and centrifuged at 500 g for 10

mm. Under these conditions, the microprecipitate pelleted, leaving

the bone marrow cells in a band at the metnizoate-medium interface.

The cells were collected, diluted to 6 x 10’ ml in fresh medium, and

0.3 ml were injected into X-irradiated (875 rad) recipient mice.

Early experiments utilizing the above protocol with calcium

phosphate coprecipitates with Cl-JO or A29 DNA were unsuccessful

(see Results). Modifications designed for cells propagated in suspen-

sion were tested using genetic transformation of CCRF-CEM cells

to MTX resistance as a model system. The optimal protocol involved

treating cells at about l0’/ml with an equal volume of calcium

phosphate microprecipitate containing DNA (20 zg/ml) or chromo-

somes (1 cell equivalent). After 15 mm, the cells were pelleted and

resuspended in 5 volumes of fresh medium (-. 106 cells/mI). Follow-

ing an overnight incubation, the cells were collected and separated

from the microprecipitates on a sodium metnizoate shelf, as

described above. These cells were then incubated in fresh medium at

2 x 105/ml for 30 hr. and MTX was added at I06 M for 72 hr.

Finally, the cells were diluted with 1.5 volumes of 0.2% agar in

RPMI + 20% horse serum and plated in culture tubes. Attempts to

genetically transform munine 4l6B cells involved an identical proto-

col, except that 4 16B cells were exposed to 3 x I 0 ‘ M MTX for 168

hr before plating in agar.

Successful adaption of the above modified protocol for genetic

transformation of bone marrow cells involved treating 108 cells in 6

ml of McCoy’s 5A medium containing 10% FCS with 6 ml of a

calcium phosphate coprecipitate with either 3T6 or L5178Y DNA.

After 15 mm, the cells were diluted into 60 ml fresh medium and

incubated for a further 4-S hr at 37#{176}C.Isolation of DNA-treated

cells and subsequent injection into recipient irradiated CBA mice

was as described above. In experiments involving mixing CBA and

CBA/T6T6 cells, 0.2 ml of LSI78Y DNA-treated cells was mixed

with 0.1 ml of 3T6-treated cells prior to injection. The MTX

treatment regimen was initiated 4 days after injection of donor

marrow cells into recipient mice. All injections were administered

intraperitoneally on a Monday/Wednesday/Friday schedule.

CFU-S Assays

The number of spleen colony-forming cells (CFU-S) in bone

marrow of experimental animals was determined by injecting 5 x

IO� or l0� cells into previously irradiated (900 rad) CBA mice.”

Spleen colonies were scored after I 0- 12 days.

DHFR Assays

DH FR activities of mouse spleens were assayed using crude

extracts prepared as follows. Spleens were minced in 50 mM Tnis,

pH 7.5, 2.5 mM MgCI2, 150 mM KCI, and 100 �ig/ml 3 mM

dithiothreitol (0.2 mI/spleen). The extract was sonicated on ice with

4 1 5-sec bursts, using a Branson sonicator at setting 5. The sonicate

was then centrifuged at I 5,000 rpm for 30 mm at 4#{176}Cin a Sorvall

refrigerated centrifuge, and the supernatant was collected and

assayed directly. DI-IFR activities were measured in a Guilford

spectrophotometer at 37#{176}C.The assay mix, in I ml, included 100

mM Tris, pH 7.5, 150 mM KCI, I 00 zg/ml NADPH, 20 �sM FH2,plus the extract and drug solution. The oxidation of NADPH was

followed at 340 nm in 4 different assays simultaneously. Spleenextracts from experimental animals were usually assayed in thepresence and absence of MTX (3 x 10’ M) in simultaneous

readings recorded on the same chart. DI-IFR activities were esti-

mated from the slopes of the 0D3,,, versus time plots. One unit of

enzyme activity is defined as a I nmole of NADPH oxidized in I

hr.

Hematocrit Measurements

Hematocnits were measured using blood drawn from the retroor-

bital sinuses of live mice using hepaninized capillary tubes.’2

Chromosome Analysis

Analysis of chromosomes from bone marrow cells of CBA and

CBA/H-T6T6 mice was based on the method of Ford.’3 Mice were

injected intrapenitoneally with 4 mg/kg colcemid 70-90 mm before

sacrifice. Bone marrow cells were flushed from the femurs of freshly

killed mice with McCoy’s SA medium, 15% FCS, and I U/mI

heparin. Cells were swollen in 75 mM KCI for 30-35 mm at 37#{176}C

and fixed in ice-cold methanol:glacial acetic acid (3:1) for I hr

(fixative changed 3 times). Chromosome spreads were prepared by

dropping the cell suspension onto microscopic slides from a height of

2-5 feet. Final chromosome spreads were stained with Trypsin-

Wright’s stain.’4 Spreads of 50-100 chromosomes were scored for

each analysis.

RESULTS

Establishment of Methotrexate Regimen

for CBA Mice

An appropriate maximally tolerated dose regimen

for MTX was first established using CBA mice. This

was done by monitoring hematocrits and CFU-S levels

in live mice as an indication of the hemopoietic sup-

pression induced by various doses of MIX. Mice were

initially X-irradiated with 875 rad and injected on the

following day with 2 x iO� bone marrow cells fromvinblastine-treated CBA donors. Injections were mi-tiated 4 days later, and hematocrits were monitored

after 3 and 6 wk. Control experiments indicated that

blood sampling (up to 0. 1 ml) from these mice, even on

a weekly basis, had no effect on hematocnits and

CFU-S values. Table 1 shows the change in hemato-

crits and CFU-S values with different dose schedules.

The schedule chosen for selection of transformed bone

marrow cells involved treatment with 2 mg/kg in the

first week following irradiation, 6 mg/kg in the second

week, and I 2 mg/kg in the third and subsequent weeks

(2/6/12 regimen). The 0.5/2/4 regimen was similar

to that employed by Cline and coworkers in their

experiments.’ However, in our hands, this regimen hadno detectable hematologic toxicity.

Failure to Transform Bone Marrow Cells to

Methotrexate Resistance Using A29 DNA

and Chromosomes

Initial attempts to genetically transform bone mar-

row cells to MTX resistance involved the use of

calcium phosphate coprecipitates with A29 DNA or

chromosomes. Chinese hamster ovary A29 cells have

been shown to overproduce a mutant species of DHFR

that is more resistant to MTX inhibition than the wild

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182 CARR ET AL.

Table 1 . Effect of Methotrexate Treatment Schedules on

Hematologic Parameters

Hematocrit HematocritDose StheduCe of MTX (mg/kg)

(Week 3) (Week 6) CFU-S/10’ Cells

Week 1 Week 2 Weeks 3-6 (%) (%) (Week 6)

0 0 0 49 51 20±3

0.5 2 4 51 50 29±2

1 4 8 51 46 4±2

2 6 12 48 27 1±1

3 8 16 39 ND’ ND

4 10 20 36 ND ND

Results are means of at least three independent measurements ( ± SD

for CFU-S measurements). Standard deviations for hematocrits were all

within 1 5% of the means. The dose schedules involved Monday/

Wednesday/Friday (p.m.) injections, and hematocnits and CFU-S levels

were assayed on Thursdays (p.m.). as described in Materials and

Methods.

‘ND. not determined due to death of mice.

type enzyme.4 DNA and chromosomes prepared from

A29 cells can transform mouse LTK cells to MTX

resistance with efficiency in excess of I0�6.8 We have

routinely used genetic transformation of LTK cells as

a positive control in our experiments. Despite success-

ful transformation of these cells to MTX resistance

using A29 DNA or chromosomes, we were initially

unable to find any evidence for transformed MTX-

resistant CBA bone marrow cells using similar proto-

cols (see Materials and Methods). This conclusion was

based on hematocrit and CFU-S measurements.

Transformation ofHuman Lymphoblast Cells in

Suspension Culture to Methotrexate Resistance

Following our failure to transform bone marrow

cells to MTX resistance with A29 DNA or chromo-

somes, we decided to examine in more detail the gene

transfer procedure. In particular, we wished to opti-

mize the genetic transformation of cells grown in

suspension culture, a procedure that we and others’5

have observed to be less efficient than transformation

of cells attached to a substrate. We observed that only

about 20% of a freshly isolated bone marrow cell

suspension adheres to the surface of tissue culture

flasks in the 4-hr period employed for exposure to

calcium phosphate-DNA coprecipitates. Most spleen

colony-forming cells are detachable in the suspension

during this periods. Attempts were made to genetically

transform human lymphoblast CCRF-CEM suspen-

sion cells to MTX resistance using A29 DNA or

chromosomes. The standard transformation protocol

used previously failed to yield MTX-resistant CCRF-

CEM. However, a modified protocol, which involved

increasing the cell number and the volume of calcium

phosphate microprecipitate by a factor of 5 (see Mate-

rials and Methods), led to the isolation, in 5 of 6 plates,

of colonies of MTX-resistant CCRF-CEM cells

induced by A29 chromosomes. The DHFR activity

associated with one of the resulting cell lines was then

characterized to test for expression of the mutant A29

DHFR species. The specific activity of DHFR in the

putatively transformed CCRF-CEM was found to be

elevated about 36-fold compared to wild type CCRF-

CEM cells (Table 2). This correlated with the

observed survival of the transformed cells at iO� M

and l0� M MTX, despite selection in 106 M MTX

(data not shown). Titration of equal amounts of

enzyme activity with different doses of MTX revealed

that DHFR from transformed CCRF-CEM cells was

more resistant to MTX inhibition than wild type

CCRF-CEM enzyme and was similar in resistance to

A29 DHFR (Fig. I). These results strongly suggest

that the MTX-resistant CCRF-CEM cells were genet-

ically transformed by donor A29 chromosomes using

the modified transformation protocol.

Failure to Transform a Biopotential Hemopoietic

Cell Line

Following the successful transformation of suspen-

sion-cultured CCRF-CEM cells, we attempted to

genetically transform the biopotential hemopoietic cell

line, 416B6 with A29 chromosomes. The cell line may

be closely related to murine hemopoietic stem cell by

virtue of a virally induced block in differentiation, and

therefore may provide a good in vitro model for

proliferating stem cells. Despite the low plating effi-

ciency (0.5%) for 4l6B cells in soft agar, we were able

to effectively screen 3.2 x I 0� cells (equivalent to 6.4 x

l0� cells plated) for genetic transformation to MTX

resistance by A29 chromosomes. However, no colonies

were isolated after exposure to 3 x iO� M MTX,

suggesting a transformation efficiency with A29 chro-

mosomes of less than 3 x 106. Other experiments

aimed at genetically transforming freshly isolated

bone marrow cells to MTX resistance, as judged by the

appea rance of MTX -resista nt gra nulocyte- macro-

phage colonies (CFU-C) in methylcellulose’6 (plating

efficiency of 0. 1%), were also unsuccessful, having

effectively screened 8 x I O� cells.

Table 2. Comparative DHFR Activities of Hamster and Human

Cell Lines

Cell Line

DHFR Activity

(U/mg Protein)

CHO 98

A29 (CHO MTX”) 4,832

CCRF-CEM 80

CCRF-CEM MTX 2.9 12

Enzyme activities are expressed in units. One unit is defined as 1 nmole

of NADPH oxidized per hour. The activities are means for 3 estimations,

with standard deviations of less than 20%.

For personal use only. by guest on December 5, 2013. bloodjournal.hematologylibrary.orgFrom

00

>‘

.� 80

C.)

a: 60LLI0

20

[MTx], M

100 �

�‘

:� \3T6- .) mouse \c-i

< spleen #{149}\a: 50U-

a�2 25

0 � i

IO� l0�� lO� 106 lO� �

[ Mlxi, M

Fig. 2. Titration of mouse spleen DHFR and 3T6 DHFR byMTX. DHFR assays were run in the absence or presence ofincreasing amounts of MTX. as described in Materials and Meth-ods.

MTX-RESISTANT MURINE BONE MARROW CELLS 183

Fig. 1 . Titration of DHFR activity for human (CEM/CEM MIX) and hamster (CHO/A29) cell lines withMTX. Enzyme activities were measured at least in

triplicate (mean shown) by monitoring the oxidation ofNADPH at 340 nm. Approximately 5 U of enzyme wereassayed for each cell line. One unit is defined as theamount of enzyme required to reduce 1 nmole ofdihydrofolate in 1 hr. (0) CEM; (�) CHO; (S) A29; (0)CEM MTX; cells transformed with A29 chromosomes.

40

Transformation ofBone Marrow Cells to

Methotrexate Resistance With 3 T6 or pHG DNA

The choice of donor A29 genetic material for pre-

vious bone marrow transformation experiments was

based on the observation that mutant A29 DHFR

genes can efficiently induce easily selectable trans-

formed MTX-resistant mouse cells.8 The previously

successful genetic transformation of mouse bone mar-

row cells by Cline and coworkers’ involved treatment

of marrow cells with DNA from 3T6 cells. Recent

studies have revealed that 3T6 cells produce a mutant

DHFR activity over 100-fold resistant to MTX inhibi-

tion compared to the wild type enzyme activity.5 This

represented a proportionally greater MTX resistance

than was found for the DHFR activities of A29 cells

compared to wild type CHO cells (15-fold; Fig. I).

Studies with the 3T6 subline, induced in this labora-

tory to grow in 2 mM MTX, revealed an even greater

relative resistance ( I 0,000-fold) of 3T6 DH FR activi-

ties to MTX (Fig. 2). The mutant enzyme activity in

this case titrated over a relatively narrow dose range of

MTX (l0��-l0 “ M) compared with the previously

characterized 3T6 activity ( l0��-l0� M).3

The presence ofone or more mutant DHFR genes in

3T6 cells might explain the success of the bone marrow

cell transformation experiments of Cline and cowork-

ers.’ Therefore, we subsequently used 3T6 DNA in

attempts to transform bone marrow cells to MTX

resistance. For these experiments, we utilized mixtures

ofsyngeneic CBA and CBA/T6T6 bone marrow cells,’

which are easily distinguishable by chromosomal anal-

ysis.’3 Mixtures of cells treated with calcium phos-

phate coprecipitates with 3T6 DNA and L5178Y

DNA in ratios of 1:2 were injected into irradiated

recipient CBA mice (Table 3). Hematocnits were

measured 43 days after injection, and the mice were

sacrified between days 46 and 58. About half of the

mice were treated with colcemid 70-90 mm before

sacrifice, and bone marrow cells from these mice were

used for chromosomal analysis. Bone marrow cells

from the remaining mice were used for CFU-S assays.

The spleens were removed from every experimental

mouse and utilized for DHFR assays. Enzyme assays

for DI-IFR were performed in the presence or absence

of 3 x 10 � M MTX for animals injected with 3T6

DNA-treated marrow cells. This dose is completely

inhibitory to wild type mouse DHFR enzyme, while it

does not inhibit the activity from 3T6 cells (Fig. 2).

A summary of results of all of the analyses per-

formed on mice injected with 3T6-treated bone mar-

row cells is provided in Table 3. In every case, animals

transformed with 3T6 DNA displayed elevated hema-

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184 CARR ET AL.

Table 3. Analysis of CBA Mice Injected With Calcium Phosphate-DNA Treated Bone Marrow Cells and Treated With MTX

for 46-58 Days

Final Percent Final

(A) DNA

Treatment

)CBA)Percent

Hematocrit

Percent 3T6

DHFR

Activity

Percent

CBA

(Marrow)

CFU-S

(per 10’ Cells)

I ± SD)

(B) DNA

Treatment

)CBA/H-T6T6)Percent

Hematocrit

3T6

DHFR

Activity

Percent

CBA

(Marrow)

CFU-S

(per iO’ Cells)

( ± SD)

None 45-53 0 (33) 30(±4) None 46-53 0 (33) 30(±4)

3T6 3T6

Mouse no. 1 34.5 39 60 Mouse no. 6 30 18 48

2 35.5 33 48 7 39.5 28 40

3

4

5

50

39.5

39

78

45

36

93

30(±17)

14(±3)

8

9

10

39

32.5

35

26

0

14

13

0

0

L5178Y 24

29

29.5

0

0

4

33

10

1(±2)

11

L5178Y

36

23.5

26

28

0

4

0

18(±9)

24

34

25

27

31

26

29.5

0 32

0

Hematocrits were measured after 43 days with blood drawn from the retroorbital sinus of donor mice. DHFR activities, measured as rates of change of

340 nm absorption, are expressed as percentage activity ratios in the presence or absence of 3 x 10 � � M MTX (from one analysis only). Activity of DHFR

in the absence of MTX was considered to be 1 00% . The ratio of CBA or CBA/H-T6T6 bone marrow cells was calculated from at least 1 00 chromosome

spreads for each analysis. This ratio is presented in the table as the percent of CBA chromosomes found of the total. CFU-S levels for each individual

mouse are expressed as means for 3-6 separate analyses.

tocrits compared to control animals injected with

untreated or L5178Y DNA-treated cells only. This

increase was striking for animal 3T6-3, which dis-

played a hematocrit in the range of uninjected con-

trols. In spleens derived from all but one animal

(3T6-9), DHFR activities characteristic of those

encoded by 3T6 genes were detected, indicating that

these genes were being expressed in the spleens of

positive animals. Chromosomal analysis of bone mar-

row cells revealed a significantly increased relative

proliferation of 3T6 DNA-treated marrow cells com-

pared to L5178Y-treated cells in all animals except

3T6-8. Finally, spleen colony-forming units derived

from marrow cells of the 3T6 series mice were detected

in all cases, except from mice 3T6-9 and 3T6-l0.

Therefore, at least two positive results suggesting

the presence of transformed MTX-resistant bone mar-

row cells were obtained for all animals, apart from

mice 3T6-9 and 3T6- I 0. These mice displayed slightly

elevated hematocrits, for reasons that are presently

unclear. The results indicate that treatment of mouse

bone marrow cells with calcium phosphate coprecipi-

tates and 3T6 DNA, using a modified protocol, gives

rise to transformed MTX-resistant cells at high effi-

ciency.

DISCUSSION

These results confirm the observations of Cline and

coworkers’ that MTX-resistant mouse bone marrow

cells can be efficiently produced in vivo by transforma-

tion with calcium phosphate-3T6 DNA coprecipitates

in vitro. Several improvements in the protocol used by

Cline and coworkers have been implemented includ-

ing: (A) exposure of more concentrated cell suspen-

sions to increased volumes ofcalcium phosphate-DNA

coprecipitates; (B) separation of these coprecipitates

from bone marrow cells prior to injection into recipient

mice; and (C) increasing the doses of MTX used for in

vivo selection of MTX-resistant transformed bone

marrow cells.

These improvements were shown to increase the

efficiencies of the genetic transformation, bone mar-

row cell administration, and bone marrow cell selection

steps in the protocol. As a result, genetically trans-

formed MTX-resistant bone marrow cells were

detected in 9/ 1 1 mice that had received 3T6-treated

marrow cells (Table 3). However, no genetically trans-

formed cells were detected in mice injected with A29

DNA-treated marrow cells or with L5178Y DNA.

The high level of resistance of the mutant DHFR

activity encoded by 3T6 DNA may have permitted

proliferation of 3T6 DNA-transformed cells in vivo in

conditions where A29 DNA-transformed cells were

unable to proliferate. In addition, in view of the

markedly altered 3T6 R400 DI-IFR gene, it is likely

that transfection with only a single 3T6 gene would

impart MTX resistance to the recipient marrow stem

cell.

We are currently attempting to isolate the cDNA

for DHFR from 3T6 cells for future gene transferexperiments. It should be possible, using the transfor-

mation procedure developed, to transfer almost any

cloned gene to mouse bone marrow cells without

impairing the proliferation capacity of these cells in

vivo. Genes coding for nonselectable markers may

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MTX-RESISTANT MURINE BONE MARROW CELLS 185

potentially be transferred to bone marrow cells with

selectable genes,”8 such as those encoding DHFR and

thymidine kinase.’9 Alternative gene transfer proce-

dures, such as cell-to-cell fusion and liposome-

mediated gene transfer, are more likely to impair the

normal functioning of these cells.

The transfer of drug-resistant genes to normal bone

marrow cells perm its i ncreased hematologic tolerance

to intensive drug treatment schedules, as demonstrated

in these studies. This tolerance may improve thera-

peutic ratios for treatment of a variety of tumors with

common anticancer agents, and experiments are

planned to assess this possibility using model tumors in

mice. A major drawback of the present method for

induction of MTX resistance in bone marrow cells is

that a relatively long selection time is necessary to

amplify MTX-resistant cells to detectable levels. Fur-

then improvements in transformation efficiencies are

therefore desirable. However, it is likely that an appre-

ciable number of bone marrow cells initially express

donor DHFR genes shortly after gene transfer, as

demonstrated in other systems.2#{176} This initial transient

expression may explain the elevated hematocrits

observed for mice 3T6-9 and 3T6- I 0 (see Table 3) in

the absence of detectable donor DHFR activity in the

spleen. Therefore, intensive chemotherapy may be

possible immediately after receiving bone marrow cells

transformed with genes concerning drug resistance.

This idea is currently being investigated.

The high efficiency of bone marrow cell transforma-

tion to MTX resistance observed in the experiments

reported supports the conclusion of Cline and cowork-

ers’ that normal munine bone marrow is as transform-

able as some tissue-cultured cell lines. Therefore, the

mouse system provides a good model system with

which to study the potential clinical applications of

bone marrow cell transformation. Further characteri-

zation of this genetic transformation system is neces-

sary prior to adaption of these procedures to humans.

The location of the 3T6 R400 DNA in the marrow

cells has not yet been determined. One explanation for

the transient resistance of mice 9 and I 0 to MTX could

be that the 3T6 DNA was extrachromosomal and not

integrated into host chromosomes.

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