tumor-associated karyotypic lesions coselected with in vitro macrophage differentiation

GENES, CHROMOSOMES d: CANCER 5:286-298 (1992)

Tumor-Associated Karyotypic Lesions Coselected With In Vitro Macrophage Differentiation

Bruce Alexander, Roland Berger, Lesley M. Day, P. Mark Hogarth, Anthony Feneziani, and Wendy D. Cook

Molecular Cytogenetics Laboratory, Department of Surgery, and Research Centre for Cancer and Transplantation, Univenity of Melbourne, Parkville, Australia (B.A., L.M.D., P.M.H., A.F.. W.D.C.); lnstitut de Genetique Moleculaire, Paris, France (R.B.)

Several cytogenetic lesions in chromosomes 2, 5. 12. and 16 have been repeatedly coselected with in vitro macrophage differentiation in a clonal murine thymic tumor cell line. Parental-type subclones, which show an extremely immature hemopoietic phenotype, do not carry the aberrations. The frequency of the stable differentiated variants is elevated by 5-azacytidine and bromodeoxyuridine, consistent with chromosome breakage being responsible for the phenotype. The frequency is also raised by dexamethasone. Since variants are 300-3,000-fold more resistant t o dexamethasone than parental clones, we interpret this t o be largely due to selection. Three of the lesions, on chromosome 2. match those previously described as associated specifically with in vivo-generated murine myeloid tumors, induced by X irradiation and corticosteroid treatment. Several implications follow from these observations. (I) In vitro differentiation in clonal tumor cell lines can be used to select for tumor-associated lesions. This should allow genetic and molecular analysis of the chromosome 2 lesions and of others that may pinpoint genes critical to macrophage differentiation and transformation. (2) Myeloid and lymphoid tumors that occur in response t o X irradiation may diverge from a common initiating tumor. (3) The hemopoietic lineage switch phenomenon, previously described by several authors, may be caused by similar o r identical chromosome aberrations. Genes Chrom Cancer 5:286-298 (1992). @ 1992 Wiley-Liss. Inc.

INTRODUCTION

Many tumorigenic cytogenetic lesions have been elucidated by virtue of their disruption of known on- congenes or of the recombinogenic lymphocyte antigen receptor genes (reviewed by Rowley, 1984). How- ever, the determination of the biochemical roles of these genes in initiation and/or progression of tumors, and of their normal counterparts in control of cell growth and differentiation, has proven much more difficult. Moreover, many well documented but not so amenable chromosomal aberrations remain unex- plained (Mitelman, 1991). Naturally this is especially common in cases of chromosomal deletions, which may span several centimorgans and many loci. Exam- ples of these are the human 5q- and murine 2 deletions, which are associated with myeloid tumors, though they are not in syntenic regions (Azumi and Sachs, 1977; Hayata et al., 1983; Fourth International Workshop on Chromosomes in Leukemia, 1984; Trakhtenbrot et al., 1988). Progress in both the identification of the genes crucial to these lesions and the analysis of their biochemical roles in tumorigenesis would be greatly aided by the development of in vitro models. Such models might ideally consist of clonal cell lines, in which chromosomal aberrations could be selected by their phenotypic effects. We present here a series of observations that together are a step towards such a model. The data shed light on known tumor-associated lesions and identify new candidate aberrations. Both types of lesions have been selected

not directly by their tumorigenic effects but by an apparent role in in vitro differentiation.

We have documented a process of macrophage differentiation occurring in a clonal murine hemopoietic cell line, which had been transformed by Abelson virus. Although the line is derived from a thymic tumor, its phenotype is extremely immature and in- cludes markers of myeloid potential. Stable macrophage-like variants of the line have been generated. In addressing the mechanism of the differentiation phenomenon, we show here that the frequency of variants is elevated by two drugs known to induce chromosome breakage, namely, 5-azacytidine (5-AC) and bromodeoxyuridine (BrdU). Accordingly, consistent chromosomal aberrations are associated with step- wise changes in the variants towards a differentiated phenotype. Furthermore, included in these aberrations are a deletion and two translocations of chromosome 2, which reflect those described previously for in vivo-generated myeloid tumors.

Aberrations of chromosome 2 are exceptionally prevalent in murine myeloid tumors induced by X irradiation and corticosteroids. After irradiation of mice of various strains, 60%) of tumors are lymphoid with thymic involvement, the remainder being longer latency but acute myeloid leukemias. Coleukemogenic

Received January 2 , 1092; rcvision acccpted April 1, 1992. Address reprint requests tn W.1). Cook, Univ. Dept. Surgery,

Royal Melbourne IIospital, Parkvillc .7050. Australia.

0 1992 WILEY-LISS. INC.

KAR YOTYPIC LESlONS AND MACROPHAGE DIFFERENTfATlON 287

treatment with corticosteroids elevates the myeloid fraction to 9(rlOO% (Resnitzky et al., 1985). Three groups have shown that deletions leading to a mini- mal loss of the D band occur specifically in 95% of the myeloid tumors (Azumi and Sachs, 1977; Hayata et al., 1983; Trakhtenbrot et al., 1988). Since both the proximal and the distal breakpoints of these lesions are highly variable, it is reasonable to assume that the critical genetic events are losses of a gene or genes. Though the simplest explanation is the common loss of a D band gene, there is no evidence arguing against the involvement of multiple loci.

Phenomena that are apparently different have been described by Cox and colleagues, who found translocations of chromosome 2 involving the C2 or E5 bands (Silver et al., 1987). Because these aberrations do not always result in detectable deletions, and since the breakpoints are so constant, it may be that activation or dysregulation of genes located in the two relevant bands is the selected event in this second class of tumors. Silver et al. (1989) have observed that interleukin (IL)lp, but not ILla, is up-regulated in tumors carrying these markers. Since these genes are located near the 2E5 breakpoint (at 2F) (DEustachio et al., 1987; Boultwood et al., 1989), the possibility has been raised that this is a direct (cis) effect of the E5 translocations on the transcription of the ILlP gene.

Deletions of chromosome 2 have been detected in preleukemic mice after irradiation and corticosteroid treatment. This has led to the conclusion that the chromosome 2 abnormalities are necessary but in- sufficient “initiating” events for myeloid leukemogenesis (Trakhtenbrot et al., 1988). Since the cytogenetic events we describe have occurred in an established tumor cell line, there are important implications for the possible roles of the chromosome 2 aberrations in the generation of the in vivo tumors. Furthermore, because the in vitro system provides the ideal control cell populations (the parent line and step- wise series of increasingly differentiated variants), a precise analysis of the association of karyotype and phenotype is possible. This should facilitate the isolation of the relevant genes and the subsequent analysis of their effects. Finally, these observations may explain the so-called lineage-switch phenomenon, in which cell lines of B or pre-B lymphoid phenotype convert to macrophages.

MATERIALS AND METHODS Cell Lines and Culture Conditions

All lines were maintained in DME medium with 10% fetal calf serum and 50 pM 2-mercaptoethanol. The induction and characteristics of the Abelson virus-induced thymic tumor cell line RB22.2 have

been described previously (Cook and Balaton, 1987). pU5.1.8 and 5774 (Rabh et al., 1976) were obtained from A. Harris (Walter and Eliza Hall Inst.). Adherent lines were passaged using trypsinization. Semiadher- ent lines were removed from tissue culture surfaces with vigorous pipetting.

Phagocytosis Assay

Cells were incubated for 16 hr at 37°C with a 50-fold excess of 3.92 pm fluorescent latex beads (Poly- sciences, Inc., Warrington, PA) and washed through a 4 ml layer of fetal calf serum before scoring on a Zeiss fluorescence microscope. A total of u500 cells were scored in each clone. Cells containing three or more internalized beads were scored as positive. For the analysis of morphology, cells were first grown on chamber slides, which in the case of RB22.2 were coated with gelatin. After incubation with the beads, slices were washed with PBS and counterstained with acridine orange. Slides were examined on a confocal laser scanning microscope (Wild Leitz) using a x 40 fluorescence oil immersion objective and were re- corded by a video printer.

Nonspecific Esterase (NSE) Staining

NSE staining was performed using a-naphthyl bu- tyrate, in a modification of a published method (Koski et al., 1976), and assessed by microscopic examination under x 200 magnification.

Extraction and Analysis of mRNA

Polyadenylated RNA from unfractionated cells was prepared, and 2 pg of RNA was loaded per track onto 1.2% agarose gels and clectrophoresed in formalde- hyde as described previously (Cook and Balaton, 1987). RNA was blotted onto Genescreen filters (Du- pont), which were hybridized and washed according to Church and Gilbert (1984).

Hybridization Probes

The FES probe was a 650-base-pair Pstl fragment of GA-FeSV proviral DNA (Hampe et al., 1982). The FMS probe was a 1.4 kb Pstl fragment of V-FMS from the McDonough strain of FeSV (Donner et al., 1982). The MYC probe was a 1.3 kb Xhol fragment cut from the pMc-myc 54 plasmid (Stanton et al., 1983). The y actin probe was a 1.6 kb BumH1 fragment of the human cDNA cut from the pHFl plasmid (Gunning et al., 1983). The TNF--a probe was a 600 bp Bull-EcoR1 fragment supplied by John Delamarter (Glaxo) and subcloned by Nick Gough (Walter and Eliza Hall Inst.). The IGp probe was a 2 kb Hind111 fragment spanning the first and second exons of the Cp region (Bernard and Gough, 1980). The TCRy

288 ALEXANDER E T AL.

probe was a 1.4 kb BamHl cDNA fragment (Yanagi et al., 1985). FCyRIII mRNA was detected with an oligonucleotide probe specific for sequences encoding the transmembrane region that is unique to this form (Hogarth et al., 1987). The ILlcl probe was a 400 bp Pstl-Xmnl cDNA fragment (Lomedico et al., 1984), and the ILI p probe was a 900 bp HindIII-PuuIl cDNA fragment supplied by R. Lang (Ludwig Institute for Cancer Research, Melbourne, Australia). All the fragment probes were labelled by the random primer ex- tension method (Feinberg and Vogelstein, 1983), and the oligonucleotide was end-labelled (Maniatis et al., 1982).

Karyotypes

Karyotypes were done using minor modifications of published methods. Cells were synchronized according to Lee et al. (1990), except that methotrexate was used at a final concentration of 3.3 x 10 - 8 M. Slides were aged for 7 days at room temperature and immersed in hydrogen peroxide for 5 min. Banding with Giemsa and Leishnlan stains was according to Leversha et a]. (1980). Slides were examined and photographed using a Leitz microscope with a x 100 oil immersion objective. At least ten photographs of each karyotype were cut and aligned.

Drug Induction of Variants

5-AC, dexamethasone (dex), BrdU, and hydroxyurea (HU) were all obtained from Sigma. Each was titrated on log-phase cells to give 10% survival at 3 days after addition. Treated and untreated cells were dispersed at limiting dilution in microtitre plates. Clones were counted and assessed for adherent/ nonadherent phenotype at 5 days post cloning. Wells were expanded for bulk culture only from plates with ten or fewer positive wells. Recloning of lines is indicated by sequential numbers separated by decimal points.

Drug Sensitivity Assays

Cells were plated at 1,000 cells per 100 pl culture in 96-well plates, in increasing concentrations of drug. At day 3 (dex) or 4 (BrdU, HU, 5-AC), cells were pulsed for 6 or 16 hr with 3TdR (0.5 pCi/well, 6.7 Ci/mmol). Cells were lysed using a sarcosyl buffer, and the radio- activity incorporated was determined by scintillation spectrometry.

RESULTS

RB22.2 was cloned from an Abelson virus-induced thymic tumor (Cook and Balaton, 1987). It was a typi- cal example of the group of thymic cell lines we have classified as so immature as to be not clearly commit-

ted to any of the hemopoietic lineages. It expressed no detectable surface markers for T cells (negative for Thy 1, CD4, CD8, IL2 receptor), B cells (negative for B220, AA4, surface Ig), myeloid cells (negative for Macl, F4/80), or dendritic cells (negative for 33D1) (unpublished results). It expressed mRNA for both of the diagnostic hemopoietic src-family kinases, HCK (B- and myeloid-specific) (Holtzman et al., 1987; Quin- trell et al., 1987; Ziegler et al., 1987) and LCK (T-specific) (March et al., 1985; Voronova et al., 1986). The status of its antigen receptor genes can be interpreted as noncommittal: no rearrangements of IGp or TCRB or expression of TCRcl or 6 were detected, but both immunoglobulin heavy chain (IGH) (D-J) and T cell

Figure I. Morphology of RB22.2 and derivatives. Cells were grown on chamber slides, incubated with fluorescent beads, washed, counterstained with acridine orange, and photographed on a confocal laser scanning microscope as described in Materials and Methods. A: RB22.2. B Stella. C a7- I. Scale bar represents I0 prn,

KARYOTYPIC LESIONS AND MACROPHAGE DIFFERENTIATION 289

TABLE I. Drug Induction of Variants

Drug Frequency“

None 1 . 1 f 0.4 5AC 8.3 f 1.7 BrdU 46 f 14 HU < 0.44 dex 26 -+ 7

aExpressed as percentage of surviving clones

receptor (TCR) y were rearranged and expressed (Cook and Balaton, 1987). Such a pattern of rearrangement and expression of receptor genes was found in other lines of this least mature class of thymic lines (Cook and Balaton, 1987) and has been previously reported in immature lymphoid as well as in some myeloid cell lines (Ha et al., 1984; Rovigatti et al., 1984; Cheng et al., 1986; Cook, 1987; Greaves et al., 1987; Ford et al., 1988).

Despite the lack of myeloid surface markers detected, RE322.2 had four other features characteristic of this lineage: It expressed mRNA for FCyRIII and low levels of mRNA for FES. It was also able to respond to the classical macrophage stimulant lipopolysaccharide (LPS) with an elevation of mRNA for tumor ne- crosis factor ( T N . a. Finally, histological staining for the macrophage enzyme NSE (van Furth et al., 1985) showed 46% of RB22.2 to be weakly positive.

Sublines Selected According to Adherence Are Differentiated Along a Macrophage Pathway

It was observed that RB22.2 (which is typically undifferentiated in its morphology, i.e., pseudospheri- cal and nonadherent) (Fig. 1A) and its parental-type and “blebby” subclones gave rise to adherent lines with a larger cell volume and a dramatically altered, spread morphology (Fig. 1R).

As is shown in Table 1, the frequency of adherent variants was increased by treating the parent with dex or with two mitotic inhibitors, 5-AC or BrdU, but not with a third, HU. From these experiments, further variant lines were established, as shown in Figure 2.

We have used several kinds of analysis to examine the differentiation states and potentials of the spontaneous and induced variants. Initially we chose to concentrate on two pairs of subclones: the “blebby” subclone .1.13 and its spontaneous adherent progeny .1.13.1.1.2, known as Stella (Fig. lB), and the 5-AC- induced semiadherent clone a7-early (a7-e) and its adherent derivative a7-late (a7-1) (Fig. lC), which arose as a dominant subclone. Southern gels showed that all

the lines had three similar viral insertions, confirming their clonal relatedness (data not shown).

Phagocytosis was assessed by the ability of cells to internalize fluorescent latex beads. This activity correlated with the degree of adherence of the cell lines, suggesting an ascending order of maturity of RR22.2 ( < 0.2% phagocytic), a7-e (O.8%), Stella (5.4%), a7-1 (28Y0).

Like the parent line, the variants were negative for T, R, and dendritic cell surface antigens when screened by Aow cytometry (data not shown). How- ever, the macrophage markers Macl and F4/80 were expressed by the most differentiated sublines. The spontaneous line Stella and the 5-AC-induced line a7-1 expressed Macl, consistent with, but not proof of, a macrophage phenotype (Springer and Unkeless, 1984). Only a subset of a7-1 expressed low levels of the F4/80 antigen, which is confined to macrophages (Hirsch et al., 1981) (data not shown).

A gradual progression from parent to the most differentiated variant was demonstrated by the nature of the NSE expression, i.e., according to the combination of the number of esterase-positive cells and the intensity of staining. The parent and a7-e contained 46% and 86%, respectively, of weakly stained cells, and Stella and a7-1 each had 65% of cells with intense and granular staining, similar tu that of the macrophage line 5774 (94% intense and granular).

Subsequently, the whole panel of variants was subjected to Northern gel analysis of mRNA for other macrophage markers. As is shown in Table 2 and Figure 3, in general the most adherent subclones showed the highest levels of macrophage-specific

RB22.2

5AC / a7e

a71 J .1 .5 --..- ..__..

.1 .13 ’.5b12, 1 A3.1 ’ /\\rdU d x 4

4 .5b2.1 .5d2.2 .5d3.1

.5d3.1.3

.13.1.1 4

Stella

3,4.

Figure 2. Derivation of variant and parental-type subclones. Each arrow represents a cloning step. Solid lines indicate selection for adherence; dotted lines, for blebbiness; dashed line, for parental (round, suspended) type.

290 ALEXANDER ET AL.

Figure 3. Northern gel analysis of macrophage marker expression. Probes used are indicated to the right of each panel, and the sizes of detected mRNAs to the left. In each case. probing of the filter with y-actin is shown below as a control for mRNA loading. Two sets of control T and macrophage-like cell lines were used. There are A3.37.4 (Cook et al.. 1987) and pU5.1 for the following blots: fms. FcyRIII, TNFcr. and ILIp. P41.1 (Cook et al.. 1987) and J774 were used for fos, IL6. ILlcr. and TCRy.

mRNAs and concomitant down-regulation of the lymphoid antigen receptor transcripts. The results shown for Stella appeared to be an exception. Though it was more adherent than its precursor, .13.1.1, its expression of some markers indicated a slight dedif- ferentiation. We believe this is due to two phenomena: cytogenetic changes (see below), which may have both deleted one copy of the IL1 locus and affected the overall stage-specific expression pattern, and phenotypic drift in culture, since the earliest cultures of this clone showed higher levels of the differentiation markers c-fes and c-fms.

Dexamethasone, 5-Azacytidine, and Bromodeoxycytidine Increase the Frequency of Variants

Since the parent line showed some lymphoid characteristics, and since myeloid cells are known to be more resistant to corticosteroids than lymphoid cells (Claman, 1972; Thompson and van Furth, 1973), the possibility existed that dex was simply selecting the spontaneous variants by virtue of their drug resist- ance. Therefore, we tested the lines for sensitivity to this drug and the other drugs. As expected, and as is shown in Table 3, none of the other drugs, 5-AC,

KARYOTYPIC LESIONS AND MACROPHAGE DIFFERENTIATION 29 I

TABLE 2. Phenotypes of RB22.2 and Variants

p t .5.1 13 13.1 13.1.1 Stella a7e a71 d2.2 d3.1 b2.1 pU5.1

frns fos FcyRlll T N F a IL-6 IL-12

TcRy Adherence”

IL-I 0 __ + + -

+/- + -

+I - + + + + S

-

+ + + +

+ + -

-

-

+ + +

+ -

+I - +/- + + +

+/-

+/- + +

-

+ + + + + + ~..

-

- + + + + + + -

-

+/- -

+/- + + + + -

- -

- -

+ + + + + + + +/-

+ / - + + + + + +

+ + + + + + + + + + + + + + + + + +I--

+ + + + +

+I - + + + + t + +

+ +

+ + +

+ +

+ +

+ + + + +

+ + + +

-

-

+ + + ND

+ + + + + + ND ND +I - ND

+ + +

aAdherence: - , suspended; + + +, requires trypsinization for complete removal from tissue culture plastic. All other values represent relative levels of mRNA. taking into account variation in loading of Northern gel tracks, as assessed by intensities of y-actin signals (see Fig. 3).

TABLE 3. D rug Sensitivity”

Mean LD50b S.D.[

5-AC RB22.2 Stella a7-I DIO.1

BrdU RB22.2 Stella a74 D I 0 . I

Hydroxyurea RB22.2 Stella a7-I DI0.I

I .76 I .59 I .55 2.2

3.3 3.3

15.0 3.0

65 41.7

I 5 7 a4

0.68-3.45 0.77-2.32 0.73-2.05

I .9-2.4

2.0-5.0 0.6-6.0 I .O-30.0 2.5-3.5

54-8 I 11-60 96-270 84-84

I .4a 0.78 0.7 I

I .5

14.2 26.7 98.2

aRB22.2 is the parent line: Stella. spontaneous variant: a7-I, adherent subclone of a7-e. variant derived after 5-AC treatment of the parent; DIO. I, subclone of D 10. variant derived after dex treatment of the parent. bDrug concentrations are expressed in pM. LD50 is the concentration required to give 50% inhibition of ’HTdR uptake. In all cases, complete inhibition was achieved within a tenfold concentration range. ‘S.D., standard deviation, shown only where data are means of three or more experiments.

BrdU, or HU, discriminated strongly between the parent and variant lines. Also as expected, each of these drugs was capable of essentially 100% inhibition of each of the lines within a ten fold range of drug concentrations (data not shown).

B y contrast, dex clearly discriminated between parental and variant clones (Fig. 4). Both €3322.2 and .5 were completely inhibited in their uptake of 3H-TdR by 30 nM dex. However, complete inhibition of each of the variant clones, whether spontaneous or arising after treatment with any of the inducing drugs, was achieved only with a 300-3,000-fold higher concentration, i.e., 10-100 pM. As is shown in Figure 4, the

shapes of the curves indicate that lower concentrations of the drug either stimulated (e.g., 5d2.2 at 10 nM) or inhibited growth (all variants between 100 nM and 10 pM), without killing the cells, consistent with their myeloid phenotype. This left open the possibility that dex was acting at least in part by selecting variants. When the combination of initial kill and lower cloning efficiency of drug-treated cells was taken into account, the incidence of variants was consistent with this interpretation; whereas RrdU-treated cultures showed a fivefold increase in variant frequency, dex-treated cultures showed no significant increase.

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ALEXANDER ET AL.

RB 22.2.5 rr;n n I--."

100.0 RB22.2 I

100 101 102 lo3 104 105 lo6 5 0 . 0 ~ 0.0

200.0

150.0 c

.5b2.3 150 .O

100.0 - 100.0

50.0 - 50.0

0.0

loo lo1 102 lo3 104 105 lo6

150.0 a7.e I 250.0

.5d2.2 200.0 T

150.0

a7.1 I 100 lo1 102 lo3 lo4 lo5 lo6

Dex conc., nM

100.0 -I I

100 101 102 lo3 104 lo5 lo6

Dex conc., nM Figure 4. Dexamethasone discriminates between parental and variant clones. Uptake of a pulse of

'HTdR on day 3 of culture in the indicated concentrations of dex. expressed as percent of untreated control cultures. Vertical bars indicate standard deviation, where this was greater than 10%. Each line represents a single experiment and each point the mean of six or I 2 replicates.

Cytogenetic Abnormalities Correlate With Macrophage Differentiation

An initial group of ten derivatives of RB22.2 was subjected to karyotypic analysis (Table 4, down to and including a7-1). Idiograms of selected chromosomes are shown in Figure 5. RB22.2 was found to be cytogenetically extremely unstable. Although the karyotype was close to diploid, in 40 cells examined no predominant karyotype was found. Loss of X and

gain or alterations of chromosomes 3,5,9, and 15 were common but not consistent. This raised the possibility that the spontaneous variants were caused by this instability, in which case all variants might be predicted to show constant karyotypic abnormalities.

This proved to be true. Although continuing instability was evident, by contrast with the parent, each of the subclones had one or more dominant marker chromosomes. In the cases of parental-type clones,


TABLE 4. Karyotypesa

RB22.2 (parent) .5 (parental) .5 bleb 2,3,4. (blebby)

Variable changes; 39-42, X, - X, + 15, + del(3)(cen : : I!+ter). 42, X, X, + 15, +del(3)(cen: :I!+ter). 42, X, X, + 15, + del(3)(cen : : I!+ ter).

42, X, - X, + 5, + 18, + del(3)(cen : : E3 +ter), + ( I 5)dic( IS; 15). 4 I , X, - X, - 14, + del(3)(cen : : E3 +ter), del(4)(cen -+A5 : : C2 + ter),

- - - - I , - - - - - - - - - - - - - - - - - . . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~ - ~ - - - - - - - - - - - - - - - - - - - - - - ~ . - - - - - - - - - - - - - - - - - - ~ - - - ~ : .I. I 3 (blebby) : . I. I 3. I. I (variant)

+del(5)(cen+CI : :E3-ter), +del(l2)(cen+B: : D I +ter), t(5;12)(cen-+D: :A1 +ter), + t(l5;3;/2)(ter+AI : : E3- iH2: :DZ-+FZ),/the same, t(l9;?)fcen+D2

44, X, - X , + 10, del(2)(cen+C2: :Fl+ter), +del(3)(cen: :E3+ter), del(l4)(cen+E2:), +2t(5;12)(cen-D: :A1 +ter), t(l2;?)(?: :?), t( l5,3)(ter+Al: :E3dF3) , t(lP;?)(cen+DZ: :?), +mar.

: .1.13.1.1.2 (variant) (Stella)

..............................................................................................

, _ _ _ _ _ - . - . - - - - - - - - . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ . . ~ . . . . - . . . ~ ~ ~ ~ . ~ . . . ~ ~ ~ ~ ~ ~ ~ ~ . . . . . . . . . . . : a7-early (variant)

~ a7-1ate (variant)

41-42, X, - X , +del(3)(cen: :I!+ter), + t(l8;?;12)(cen+E4: : ? : :A1 +ter),/the same, 2 del( I 2) (cen + D I :) .

41, X, - X , + 15, +del(3)(cen: :I!+ter), +t(/3;2)(cen+D2.2: :E5+ter), + t(16;12)(cen+C3.3: :A1 +ter),/the same, t(5;13)(cen-G2: :D2+A5).

~~-~ .........................................................................................

.5. I (variant)

.5d2.2 (variant)

.5b2. I (variant)

~ .5d3.1 (variant)

I .5d3. I .3 (variant)

42, X, - X , + IS, +del(3)(cen: :I!+ter), del(5)(cen+E5:), +t(l,5)(cen+E: :I!+ter),

39, X, - X , del(3)(cen: :B+ter), +t(?;5)(?: :B+ter), +t(I5;16;I2)(cen+E: :C3+6I : :A1 +D). 42, X, - X , + 12, + 19, del(3)(cen: :B+ter), +t(?;5)(?: :I!+ter), t(l6;?)(cen+C3: :?).

42, X, - X, + 9, del(3)(cen : : I!+ ter), del(4)(cen + C I : : C6 + ter), +t(l3,12)(cen+D2: :A1 +ter), +mar l , +mor2.

41-42, X, - X , +9, del(3)(cen: :I!+ter), del(4)(cen+CI : :C6+ter), t(Z;IO)(cen+E5: : C I +ter), +t(13;12)(cen+D2: :A l - t ter ) , +mar l , +mar2./the same, +t(X;l2)(cen+ter: :A1 +ter).

t( I l ;3 / I2/?)(cen +E2 : ?).

..............................................................................................

: {

. ........~~~.~_ ^ _ ^ . . . . . . . . . . . . . . . . . . . . . . . . ~ . ~ ~ ~ ~ ~ - ~ ~ - ~ ~ ~ . . . . . . . . . ~ . ~ ~ ~ ~ . . . . . . . . . . . . . . . ~ ~ ~ - ~ ~ ~ .

aBoxes enclose sequentially derived sublines (see Fig. 2).

these had been detected as inconsistent changes in the parent. For example, subclone .5 bore two constant markers, trisomy 15, and trisomy 3 with a deletion (Al-3) in one copy. Importantly, its three blebby but otherwise parental-type subclones shared this exact karyotype. The gain of a deleted 3 was also consist- ently expressed in all the variants.

However, other changes stood out as potentially related to the variant phenotype. These were the loss of one copy of X and the presence of translocations and deletions. The first of the translocations involved band 12A1 and occurred in both the spontaneous and 5-AC-induced groups of variants (Fig. 5D). The spontaneous series also showed reduplication and translocation of chromosome 5 (Fig. 5C). In the last stage of each series, aberrations of chromosome 2 appeared. In a7-1, this was a translocation product joining 13D2.2 and 2E5, whereas Stella showed a deletion of 2CSE5 (Fig. 5 4 .

Despite the consistent features of the variant karyotypes, they tended to be obscured by other aberrations. We reasoned that this complexity was a result of relatively long periods (months) in culture. There- fore, we analyzed karyotypes from four more variants from the .5 subline, one spontaneous (.5.1) and three

druginduced (5d2.2, .5d3.1, and .5b2.1), as soon as possible after selection (within 1 month). The phenotypes of these variants are included in Table 4. Note that .5.1 showed no macrophage differentiation (Table 2), despite its adherent morphology.

As expected, the karyotypes of these clones were simpler. Again, the dominant changes were translocations into band 12A1 (Fig. 5D). This band was not affected in subline .5.1. In addition, translocations onto chromosome 5, band B, appeared three times, including in .5.1 (Fig. 5C). Band 16C3, which had been translocated in a7-1, was affected in two more of these variants (Fig. 5B).

Both the chromosome 2 abnormalities described above occurred in the last, most differentiated subclones of their series. Therefore, we attempted to re- produce this phenomenon by selecting for further differentiation in the variant .5d3.1. Two more adherent subclones were analyzed: One was tetraploid [a common phenomenon in macrophage cell lines (Mat- sushime et al., 1991)], while the only consistent change in the other line was indeed a translocation into chromosome 2, band E5. In this case, the translocation partner was chromosome 10 (see Figs. 5A and 6).

CHROMOSOME 2 A

a7LATE STELLA D3.1.3

13

E5 i

CHROMOSOME 16 D2.2

TE 12! I 82-1 a7LATE

n

CHROMOSOME 5 u

..................................................... , .5B2.1 .5D2.2 __

,

' T n t / I 13 13.1.1 STELLA

L . ...............................................

-5.1

I D

..................................

a7EARLY a7LATE

CHROMOSOME 12 D2.2 .................................... m-

j .13.1.1

STELLA D3.1 03-ls3

L.- ....................................

Figure 5

m w o T r P i c LESIONS AND MACROPHAGE DIFFERENTIATION 295

DISCUSSION

In Vitro Cell Differentiation Can Be Associated With Karyotypic Aberrations

We have demonstrated here a close association between certain cytogenetic abnormalities and in vitro macrophage differentiation in subclones of RR22.2. In all five parental-type subclones, the only aberrations seen had been detected in the parent line and were clearly clonally inherited. In striking contrast, each of the differentiated subclones carried at least one new aberration. Moreover, four chromosomes, 2,5,12, and 16, were repeatedly involved.

Variants Are Induced by Drugs That Affect Fragile Sites

The fact that both 5-AC and BrdU were able to elevate the frequency of the differentiated variants, in a line that was already karyotypically unstable, rein- forces the cytogenetic evidence that both the spontaneous and the induced variants arose as a result of chromosomal breakage. This is the only mechanism documented to be common to 5-AC and BrdU (Harri- son et al., 1983; Hori, 1983). However, the two drugs do each have multiple actions. The most noteworthy in this context is the demethylation of DNA and associated induction of transcription by 5-AC (Taylor and Jones, 1979; Lassar et al., 1986). We cannot exclude this as the mechanism of generation of a7-e. This issue may be clarified by experiments, which are underway, to determine whether X irradiation also mediates the effect.

The drug induction pattern also raises the possibility of an association with fragile sites, overlapping subsets of which have been shown to be induced by both 5-AC and BrdU (Hecht et al., 1988) but not by HU. These sites are known to be recombinogenic. A total of 38 fragile sites have been mapped in murine cells (Djalali et al., 1987; Elder and Robinson, 1989). Three of these, 2E5-F, 5B, and 13D2, coincide approxi- mately with recurring lesions detected here. The evidence from the literature concerning fragile sites in human chromosomes indicates they are not the sites of tumorigenic lesions (Sutherland and Simmers, 1988). Nevertheless, the drug-induction data pre- sented here suggest strongly at least that a common mechanism is involved. Moreover. studies on chromo-

Figure 5. Representative idiograms. Only the chromosomes involved in recurring events are shown. For each relevant subclone (indicated above the idiograms). all copies of the indicated chromosome are represented. Breakpoint bands of the principal chromosome are indicated. Breaks are shown at the lower edge of the observed breakpoint bands, regardless of the estimated positions of breaks within them. Boxes enclose aberrations occurring in sequentially related sublines. Banding patterns are taken from Lyon and Kirby (1991).

Figure 6. Chromosome 2 abnormalities. For each of the relevant variants, normal and aberrant copies of chromosome 2 and its translocation partners are shown.

some 2 lesions induced by radiation support this sug- gestion (Breckon et al., 1991).

Three of the Aberrations Are Similar to Those Found in Myeloid In Vivo Tumors and Thought to Be Contributing to Their Initiation

In each of the series of differentiated variants described here, the last step is associated with an aberration of chromosome 2. We are assessing the possible roles in these lesions of several candidate genes. Given the evidence from in vivo-derived tumors, the relative importance of gene deletions vs. breakpoint events in each of these variants cannot be predicted.

Our data tend to exclude a possible role for IL-10. Fuhlbrigge et al. (1987) have reported that expression of IL1 CY and -p genes is induced in normal macrophages during differentiation stimulated by adherence to tissue culture plastic. Consistent with this is our finding that IL-1 p and to a lesser extent IL-1 a are up-regulated gradually in the course of macrophage differentiation in our variants, peaking in five of the

296 ALEXANDER ET AL.

most differentiated clones, three of which do not show chromosome 2 anomalies. The in vivo translocations described by Silver et al. (1991) may differ genetically from the a7-1 lesion. However, these data certainly imply an explanation for their results, especially in the light of their recent report that 800 kb of DNA surrounding the IL-1 p gene is undisrupted (Silver et al., 1991).

Other Important Similarities Between the In Vivo System and the Current In Vitro Chromosome 2 Phenomena Suggest That They Are Mechanistically Related

1. Myeloid tumor cells appear in both cases in association with thymic-derived tumors. In the current report, the myeloid clones derive directly from a thymic tumor line. Such a direct relationship for the in vivo tumors would be difficult to demonstrate. How- ever, after X irradiation of intact mice, where thymic tumors fail to appear, myeloid tumors generally arise (Upton et al., 1958).

2. Cortisone favors the appearance of the myeloid tumors (Resnitzky et al., 1985). Similarly, the in vitro myeloid variants are selected by a cortisone analogue, though this does not select exclusively for the chromosome 2 anomalies.

3. Loss of chromosome X occurs in all the in vitro variants and in only one of the parental-type clones. Losses of X and Y were also noted to occur, among other anomalies, with the chromosome 2 aberrations in the in vivo tumors (Hayata et al., 1983; Resnitzky et al., 1985; Silver et al., 1988).

Novel Karyotypic Lesions Are Also Associated With Differentiation

The predominant novel aberrations observed are translocations into bands 5B, 12A1, and 16C3. In our attempts to correlate the phenotypic and karyotypic data, subclone .5.1 should be very informative. Since its only nonparental phenotypic marker is adherence, it may allow us to dissect genetic events affecting this property from more broadly acting regulatory events.

Both 5B and 12A1 participate in recurrent translocations involving various partner chromosomes, even within single clonal “lineages,” e.g., a7-e and a7-1. These are reminiscent of so-called jumping translocations, which have been identified, though rarely, in primary human tumors (Reis et al., 1991). Their apparent frequency in this system may be explained by the presence of a fragile site and a nucleolar organizer region at 5B and 12A1, respectively (Dev et al., 1977; Davisson et al., 1981).

It is striking that, in all these cases, the translocations are unbalanced, and the same derivative is re-

tained. For the events at 5B and 12A1, this results in partial tri- or tetrasomy of the chromosome (see Fig. 5). It may be important that, as is shown in Figure 5, apparently simple trisomy 5 and 12 was also observed. We note that this contrasts with a previous report in which presumed mutants gaining respon- siveness to a macrophage differentiation signal had lost one copy of chromosome 12 (Azumi and Sachs, 1977).

Each Step of In Vitro Differentiation May Result From One or More Genetic Events

The events detected as karyotypic alterations could confer the myeloid differentiated phenotype without preventing continued growth of the cells. Alterna- tively, in an indirect mechanism, a first event may induce myeloid differentiation, which would normally be terminal, i.e., would extinguish tumorigenicity and in vitro growth. In this case, a second event “rescues” the differentiated cells to growth and detection in our system, as proposed by Klinken et al. (1988). Further- more, each of these steps could in principle result from cooperation between multiple genetic events. It will be of considerable interest to determine which of these models pertains in this system and, if it is the latter, which types of event (if not both) can be caused by the chromosomal alterations.

The roles of the chromosome 2 lesions in these models pose an interesting question. A rescue role would reconcile their selection in both the in vivo and the in vitro systems. We are currently assessing whether the chromosome 2 variant lines show altered growth or in vivo migration patterns that might explain the selection for the chromosome 2 lesions in the in vivo tumors.

The Mechanism May Explain the Phenomenon of Hemopoietic Lineage Switching

This was originally described by Boyd and Schrader (1982) as the generation of macrophage-like cells from the A-MuLV transformed pre-B cell line ABLS 8 after treatment with 5-AC. No evidence for the mechanism invoived in that system was gathered. Several other groups have since reported either spontaneous or induced switching of T or pre-B tumors or cell lines to macrophage phenotypes (Hershfield, 1984; Holmes et al., 1986; Davidson et al., 1988; Hanecak et al., 1989). In one example, macrophages arose from pre-B and B cells expressing v-raf and either v-myc or c-myc (Klinken et al., 1988). In that study, karyotypic instability was documented, though detailed banded karyotypes were not performed. The common features of karyotypic instability and induction by 5-AC argue that lineage switching and the differentiation


events described in this study (which may in fact represent a version of switching from pre-T to myeloid phenotypes) occur through similar mechanisms.

In summary, we have shown that biologically and oncologically relevant karyotypic changes occur spontaneously, can be induced at high frequency, and can be coselected with differentiation events in a clonal tumor cell line in vitro. In principle, these observations should be applicable to other tumor cell types. The many advantages of an in vitro system should render possible the initial identification, followed by the analysis of the functions, of the pivotal genes affected by many tumor-specific chromosomal abnormalities.

ACKNOWLEDGMENTS

This work was supported in its early stages by the Ludwig Institute for Cancer Research and more re- cently by grants from NH&MRC (Aust.), The Anti- Cancer Council of Victoria, and The William Buck- land Foundation. L.M.D. was a University of Melbourne McFarlane Rurnet scholar. We are grateful to Mark Holloway and Ewa Witort for excellent assistance; to Margaret Leversha, Howard Slater, and Anne Robertson of the Royal Children’s Hospital, Mel- bourne, for initial help with karyotypes; and to Dr. Rob James of the Melbourne branch of the Ludwig Institute for thoughtful discussions and for reading the manuscript. We owe a special debt to Pierre Smith, with the assistance of Ben Kreunen and Lind- say Cox, for expert and patient advice and help with all aspects of the photography and artwork.

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