Loss of clonogenicity in agar by differentiating erythroleukemic cells

Pergamon Press

Life Sciences Vol. 16, pp . 1241-1252 Printed is the II .S .A.

LOSS OF CLOL~OGBNICITY IN AGAR BY DIFFBRBNTIATING BRYTBROLEUICBMIC CBLLS~ Harvey D . Preisler, ** Joha D . Lutton, Miriam Giladi, xeaaeth Goldeteia, and Bsmail D . Zanjani Departsaat o! Medicine A, Rosvell Park Memorial Institute, Hn!lalo, New York, and Departsaat of Physiology, Monat Sinai School o! Medicine, New York, N .Y . (Received is final form March 6, 1975) Bummary inclusion o! DMSO is the culture media o! Friend erythrolaukemia calls temporarily interferes with the initiation o! DNA synthesis which occurs when these cells are seeded in fresh growth aedium . By 48 hours of culture sore than 90 " of the cells in the DMSO-containing cultures have initiated DNA synthesis . As a result o! the inoreasiaq proportion of di!lereatiatiaq cells in the culture, colony-forming etlicisncy in colt agar by these erythrolankaaic oelis declines by 99 " during 4 days of culture in the presence o! DM80 . Less than 1~ o! muriae arythrolaukeaic cells growing is suspension culture differentiate spontaneously along the srythroid pathway .

The addition of dimethyl sulloxide (DMSO)

to the culture medium iaareases the proportion of differentiating calls to 40t attar 4 days of growth (1) .

lihile dilfarsntiatiaq,

these erythroleukemic calls undergo significant biocheaical and morphologic chanq® .

Ths diflereatiatinq cells synthesize

Supported by USPHS Grants 1 RO1-AM5-15525-03, matioaal Institute of Arthritis and Metabolic Diseases, sad CA-12737, National Cancer Institute . Scholar of the Leukemia society o! Amerioa 1241

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globin mRNA (2,3) globfn (4) and become responsive to erythropoietin (5,6) . We now wish to report our studies on the formation of colonies by these erythroleukemic cells in soft agar . results demonstrate that : 1)

The

these cells form only undiffer

entiated colonies, the number of which are increased by the addition of conditioned medium rich in CSF ;

2) when differ-

entlatlon of these cells is induced by DMSO, there is a decline in colony forming ability,

3) the decline in clonogenicity in

agar occurs while the cell numbers in DMSO-containing suspension cultures is increasing exponentially and more than 90t of the cells are synthesizing DNA . Methods Cells :

Friend erythroleukemia cells (clone ~745A) were

cultured in the presence or absence of DMSO (2i v/v) for 4 days as previously reported (1) .

An aliquot was removed

daily for cell counts and slides were made using a cytocentrifuga and stained with benzidine according to the method of Staphenaon (7) .

The slides were counterstained with Wright-

Giemsa and the proportion of benzidine positive cells (H+) determined by one observor who classified 500 cells in a single blinded fashion . RadioautocLraphic Studies : To determine changes in labeling index during culture, aliquots of cells were removed from control and DMSO-containing cultures each day and reauapended in 1 ml of fresh medium at a concentration of 1 x 10 6 cells/ml and incubated for 30 min . with 0 .5 uCi of 3H-TdR .

After 24 hours of culture, 4 cc

of cells were removed from control and DMSO-containing cultures, placed into small bottles and 0 .2 uCi of 3H-TdR was added .

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Twenty-four hours later, the cells were harvested and slides made . After the appropriate fluration of incubation with 3B-TdR, 5 ml of cold tissue culture media containing SOY TdR/ml (sol T) was added and the cell suspension was centrifuged, the auper aatant discarded and the cells resuspended in sol T and elides were made .

The slides were washed twice is absolute methanol

for 10 minutes and than left in cold running water overnight . The slides were dipped in Rodak NTB emulsion and developed after 14 days .

Cells with more than 5 grains located over their

nucleus ware considered to be labeled .

Each experiment was done

twice with a minimua o! 4 slides counted for each experimental point and 1500 cells counted per slide . Soft Agar Studies : 1.

Plating : The erythroleukemic cells were suspended in agar-medium

using procedures siailar to those previously described (8,9) . The agar-medium consisted of 0 .3 " agar (Difco) in McCoy's 5A tisane culture mediua supplemented with extra aaino acids, vitamins, pyruvate, glutaaine, serine, asparagine sad sodium bicarbonate (Grand Island Biological Co .) .

Fetal calf serum

(Flow Labs) was added to a final concentration of 15 " v/v and bovine serum albumin (Miles Labs), prepared according to thn method of

Norton, et al . (10),

concentration of 10 " v/v .

vas added to a final

This preparation was then aaintaiaed

at 37oC is a water bath until addition of the erythrolnukemic cells, after which 1 ml was pipstted into 35 x 10 ma Falcon culture dishes with or without coaditionad mediua (with known CSF activity) .

The plates were peraitted to get at rooa

temperature, and were incubated at 37oC is a huaidifiad ataos-

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Erythroleukemic Cell Colonies

phare coneiating of 7a C0 2 and balance air .

Vol . 16, No . 8 Each experimental

point consisted of 5-6 plates . Conditioned medium was obtained from mouse L-cells grown in NCTC 135 serum free medium (Grand Island Biological Co .) . A standard dose of 0 .10 ml of this aedium stimulates the formation of 93 + 10 colonies per 10 5 Swiss Webstar mouse bone marrow cells .

After appropriate culture dishes were inoculated

with 0 .10 ml conditioned medium, the cell agar-mediua preparation was added and the culture dish swirled to ensure adequate mixing of the conditioned medium with the cell suspension,. 2.

Co lony Sçorin~ The number of colonies was determined after incubation

for 6 days by counting the number of colonies/plate using a tissue culture inverted microscope with a net reticule grid (400 sqn)

in one ocular .

group of 40-50 cells .

A colony was defined as a discrete

When large numbers of colonies were

obtained, representative areas of a plate were counted and an average for the entire plate determined .

i n preliminary

trials, it wan found that estimates of colony numbers were within 103 of the actual numbers . were made for each plate .

Three counts and/or estimates

For morphologic studies, colonies

were removed frog the agar with a Pasteur pipette, placed on a slide sad stained for peroxidase

(11) .

Results Inclusion of DMSO in the culture media of erythrolaukemia cells results in a slowing of the growth rate of the culture . Figure 1 illustrates that the initial rate of increase is cell number in control culture was greater than that in DMSOcontaining cultures .

However, the nuabar of cells prenant in

each culture wan equal after 4 days of culture .

Radioauto-

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graphic studies o! cells which were pulse labeled !or 30 min . with 3H-TdR demonstrated that the proportion o! cells aynthesisinq DNA in control cultures was aazimal within 24 hours of seeding (Fig . 2) .

For DMSO containing cultures, however,

the proportion of DNA synthesising calls was maximal 48 hours after seeding .

when 3H-TdR was ooatinuoualy present between

the 24th and 48th hours of culture, 98s of the cells in the control cultures and 92s o! the cells in DMSO-containing cultures had become labeled .

FIG . 1 Growth curve for control cultures and cultures containing DMSO . In all cases more than 99t of the cells e:eluded trypan blue . The number of colonies formed in soft agar was directly related to the number o! erythroleukenic cells seeded (Table 1) . The colonies reached maximum aiss at 6-T days after seeding and consisted o! a tight cluster of undifferentiated mononuclear cello which did not contain perozidase-positive granules .

Inclusioa .of conditioned medium resulted in an

increase in colony-forming efficiency . There was no significant change in the proportion of colony-forming calls present is suspeaeioa cultures o! control

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!0~

60

0

y~ HOURS OF CULTURE FIG . 2

Percentage of cells which incorporated 3 $-TdR during a 30 min . exposure to the isotope . Results are expressed as mean + S .E . TAHLE 1 Colony Formation by Erythroleukemic Cells Seeded at Different Concentrations With or Without the Addition of Conditioned Medium . Cells were Taken from 2-Day Old Suspension Cultures aad Plated in Agar Culture . Counts Represent M + SE for 5 Plates . Number of Cells Seeded 10 2

10 3

5 x 10 3

Number of Colonies per Plate Control Cells

13 + 4

140 + 20

695 + 75

Cells + Conditioned Medium

20 _+ 3

234 _+ 30

1010 _+ 120

cells grown for 1-4 days prior to assay of colony forming ability .

Hy contrast, growth in the presence of DMSO was

associated with a marked decline in the proportion of colony-

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Vol. 16, No . 8 foraiag sells (Fig . 3) .

Culture in the presence of

DMSO

for

as little as one day was associated with a significant decline in colony-forming ability despite the absence of a detectable increase in the proportion of benaidina positive celle in these cultures (Fig . 3) . of

DMSO,

cultures .

lifter 4 days growth in the presence

colony-loaning efficiency was only is that of control The coloaias which did form were similar in size,

growth oharacteriatica, and cellular composition to those which were derived from cells of control suspension cultures .

FIG.

3

Effect of inclusion of DMSO in the suspension culture medium oa the proportion of beazidine positive cells in the culture and on the clonogenicity of these cells in agar . The addition of conditioned medium also appeared to result in an increase in the colony-forcing efficiency of cells frog days 1,2, and

3-DMSO

containing cultures (Table 2) .

However, the total number of colonies formed even in the presence of conditioned median was significaatly less than control cells .

Colony-forming efficiency of cslla cultured

in the presence of

DMSO

for 4 days appeared to ba stimulated

by conditioned madina, but the nuabar of colonies were too low to parait a mnaniagfnl comparison (Table 2) .

The addition

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of the conditioned medium to control as well as DMSO-treated cells did not result in the formation of myeloid differentiated colonies . TABLE 2 Colony Formation by DMSO Treated Erythroleukemic Cells Grown in Suspension Culture for 1-4 Daye and then Plated in Agar Culture With or Without Conditioned Medium . 5 x 10 3 Cells were Seeded per Plate . Counts Represent M + SE for 6 Platen . Duration of Growth in Suspension Culture (Days) 4 Number of Colonies Per Plate DMSO Calls Alone

525 + 51

196 + 21

57 + 7

DMSO Celle + Conditioned Medium

796 _+ 60

301 _+ 45

110 _+ 15

Discussion Previous studies have demonstrated that the addition of DMSO to suspension cultures of erythroleukemia cells results in a lag in both cell growth (1) and in the rate of increase in DNA synthesis which occurs after seeding the cells in fresh media (12) .

The present studies demonstrate that both

of these phenouena occur bncause DMSO temporarily prevents the majority of cells from initiating DNA synthesis after seeding in fresh media .

ey the 48th hour after seeding,

however, more than 90i of the cells in the DMSO-containing cultures had initiated DNA synthesis . These erythroleukemia cells have been cloned in soft agar (13) .

The colonies formed consist of undifferentiated

calls and the inclusion of L cell conditioned median is the agar increases the efficiency of colony formation .

The colony

forming efficiency of the culture progressively declined

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during culture in the presence of DMSO .

Two explanations of

this phenomenon suggest themselvess 1) a decline in clonogenicity in agar resulting from the increasing proportions of differentiating cells in the culture or 2) a nonspecific tozic effect of DMSO .

Several factors favor a relationship

between the induction of differentiation and the lose of clonogenicity .

Tha soft agar system does not support the formation

of erythroid colonies by normal bone marrow cells (7) . Similarly, while DMSO-treated erythroleukemic cells can fore erythroid colonies in plasma clots (14), they were unable to do so in soft agar .

Furthermore, once the process of DMSO-induced

differentiation is begun, the process of differentiation is irreversible, with individual cells continuing to differentiate even after removal of DMSO frog the culture median (15) .

Takes

together, these data suggest that the progressive decline in colony forming ability by DMSO-containing cultures is a rsElection of the increasing proportion of differentiating

oe us in the culture .

On the other hand, it is possible that the progressive loss of clonogeaicity in agar is a result of a to :ic effect of DMSO on the cloaoganic cells is the culture .

The initial lag

is cell replication in the DM80-containing cultures may ba a reflection o! such toxicity .

If this warn the case then the

progressive decline is clonogenicity might be the result of a cumulative tozic effect . this is not the case .

Several observations sugqeat that

More than 90 " of the suspension culture

cells were synthesiaiaq DHA at a tine when the colony lorainq efficiency was declining by 2S folfl (between 24 and 48 hours o! culture) .

Similarly, this decline is clonogenicity ocourrad

during as ezpoaential increase is the nuabar of calls in the

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culture and at a time when more than 99i of the cells excluded trypan blue .

However, these factors do not conclusively rule

out a toxic cause of decreased clonogenicity since supralethally damaged cells can in some cases synthesize DNA and qo through several divisions prior to cell death . The lose of clonogenicity in vitro during growth in suspension culture in the presence of DMSO provides an in vitro parallel of the observation that erythroleukemic cells cultured in the presence of DMSO are less leukemogenic than cells cultured in the absence of DMSO (1) .

While these studies

suggest that a DMSO alteration in immunogenicity of the cultured cells does not play a role in their decreased malignant potential, it is not yet possible to conclusively ascribe the altered malignant potential of DMSO-treated cells to the increase in the proportion of differentiated cells in the culture . References 1.

C . FRIEND, W . SCHER, J .G . HOLLAND, and T . SATO . Nat . Aced . Sci . 68, 378 (1971) .

2.

J . ROSS, Y . IXAWA, and P . LEDER . 69, 3620 (1972) .

3.

H .D . PREISLER, W . SCHER, and C . FRIEND . 1, 27 (1973) .

4.

H .D . PREISLER, D . HOUSMAN, W . SCHER, and C . FRIEND . Nat . Aced . Sci . 70, 2956 (1973) .

5.

H .D . PREISLER and E . ZANJANI . (1973) .

6.

H .D . PREISLER and M . GILADI .

7.

J .R . STEPHENSON, A .A . AXESRAD, D .L . McLEOD, and M .M . SHRSSVE . Proc . Nat . Aced . Sci . 68, 1542 (1971) .

8.

W .A . ROHINSON, and B . PIxE . Hemopoietic Cellular Proliferation, p . 249, Grune and Stratton, New York (1970) .

9.

D . METCALF and M . MOORE, Haemopoietic Cells 24, 123, Am . 8lsavier, New York (1971) .

Proc .

Proc . Nat . Acad .Sci . Differentiation Proc .

Blood (abstract) 42, 1017 Nature 251, 645 (1974) .

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10 .

R .G . WORTON, E .A . McCDLLOCH, and J .E . TILL . J . Cell . Physiol . 74,171 (1969) .

11 .

L .S . KAPLOfi .

12 .

C . FRIEND, W . SCHER, H .D . PREISLER, and J .G . HOLLAND . Unifying Concepts of Leukemia . Proc . V Int . Symp . Comp . Leukemia Research, Padova/Venice, 1971 . Bibl . Haemat ., no . 39, ed . R .M . butcher, Kargen, Hasel (1972) .

13 .

M .C . PATULEIA and C . FRIEND .

14 .

K . GOLDSTEIN, J .D . LUTTON, H .D . PREISLER, sad E .D . ZANJANI . Blood, in press (1974) .

15 .

H .D . PREISLER sad M . GILADI . (1974) .

Blood 26, 215 (1974) .

Cancer Res . 27, 726 (1967) .

J . Cell . Physiol ., in press,