bcr-abl-Induced Cell Lines Can Switch From Mast Cell to - Blood

bcr-abl-Induced Cell Lines Can Switch From Mast Cell to Erythroid or Myeloid Differentiation In Vitro

By Andrew G. Elefanty and Suzanne Cory

The chimericbcr-ablgene formed by the Philadelphia translocation is thought to initiate chronic myeloid leukemia. Engraft- ment of mice with bone marrow cells infected with a bcr-ab1 retrovirus has been shown to elicit multiple hematopoietic disorders, including a clonal but nontransplantable hyperpro- liferation of erythroid and/or mast cells. Culture of spleen and bone marrow cells from such mice usually yielded mast cell lines, even when erythroid disease dominated the primary animal. The mast cells, which carried the same proviral insert as the primary disease, generally grew slowly and were neither transplantable nor clonogenic in agar until they had been cultured for several months. Unexpectedly, several bcr-ab/-induced lines switched in vitro from mast cell to

HRONIC MYELOID leukemia (CML) is a clonal C stem cell malignancy' typically distinguished by the Philadelphia chromosome (Ph')? a shortened chromosome 22 that results from a reciprocal 9;22 transl~cation.'.~ The translocation juxtaposes the N-terminal region of the bcr gene (on chromosome 22) and the body of the c-ab1 proto-oncogene (from chromosome 9), thereby creating a hybrid bcr-ab1 gene encoding a 210-Kd fusion polypeptide? The bcr-ab1 gene created by a variant form of the translocation confined to Ph'-positive acute lymphoblastic leukemia (ALL) involves a smaller bcr moiety and encodes a polypeptide of 190 Kd.6 The heightened tyrosine kinase activity of the bcr-ab1 proteins7 is believed to be the key feature inducing disease.

The chronic phase of CML, which can be regarded as a preleukemia, initially exhibits an excess of maturing myeloid elements in the blood and hematopoietic organs. After 3 to 5 years the disease accelerates and eventually culminates in a fatal blast crisis, usually an acute myeloid or lymphoid leukemia, although erythroid, megakaryoblastic, and basophilic variants occur infrequently.'~~ The inability of chronic-phase CML cells to be maintained in culture has limited analysis of the events involved in progression to blast crisis. The existing CML cell lines, in fact, derive from patients in the terminal blast phase."

Early studies of the effect of expressing a bcr-ab1 cDNA" in hematopoietic cells established that immortalised cell lines were relieved of their previous growth factor require- ment and rendered tumorigeni~.'~"~ Low-level growth factor production by one such line raised the possibility that ab1 oncogenes facilitate establishment of an autocrine l00p.'~ Most attempts to mimic CML, either in vitro by infecting long-term murine bone marrow cultures with a bcr-ab1 re t rovi r~s , '~~ '~ or in vivo through bcr-ab1 transgenic

yielded only lymphoid disease. A more promising approach has been to transplant lethally irradiated mice with bone marrow cells infected with a bcr-ab1 retrovirus. A high incidence of hematopoietic disease was o b s e ~ e d . ' ~ - ~ ' A few mice exhibited the granulocytosis expected of CML:0321 but most developed macrophage, erythroid, mast cell, or lymphoid tumors.

This report focuses on mast cell lines established from mice with erythroid and/or mast cell disease. Many were

megakaryocytic and/or erythroid character, and one became myeloid. The dramatic phenotypic shifts seem likely to involve changes occurring within progenitor cells maintain- ing the clone, rather than mutation of mature mast cells. The variant lines exhibited substantial spontaneous differentiation, despite being readily transplantable and therefore fully transformed. The production of hematopoietic growth factors by the mast cell lines and their phenotypic variants may implicate an autocrine loop in their evolution. These novel bcr-ab/ cell lines should aid in the study of genetic events in the progression from chronic to acute leukemia and facilitate analysis of hematopoietic lineage commitment. o 1992 by The American Society of Hematology.

not malignant until they had been cultivated in vitro for several months. Surprisingly, several clonal bcr-abl-expressing cell lines switched in vitro from mast cell to erythroid or myeloid differentiation. The implications of these findings for CML are discussed and hypotheses to explain the "lineage switching" proposed.

MATERIALS AND METHODS

Establishment of Cell Lines Bone marrow or spleen cell suspensions were seeded at 1 to 2 x

lo6 cells/mL in 10 mL Dulbecco's modified Eagle's medium containing 20% fetal calf serum and 0.05 mmol/L 2-mercaptoetha- no1 in 80 cm2 flasks (Nunc, Kamstrup, Denmark) and incubated at 37°C in a humidified atmosphere of 10% CO, in air. After 5 to 7 days, or sooner if cell proliferation was rapid, 5 to 10 mL of fresh medium was added. Thereafter, half medium changes were performed at least weekly. Cell lines that were 'slow' growing had doubling times of 2 to 7 days while 'fast' growing cell lines doubled every 1 to 2 days. Cell lines were frozen and stored in liquid nitrogen at regular intervals, especially after phenotypic 'switching,' to circumvent the problem of dedifferentiation that occurred with continuous culture of these lines.

Analysis of DNA and RNA

Nucleic acid isolation and fractionation was as described previ- o ~ s l y . ~ The probes used for blot analysis included the following: IgE receptor, the 1,036-bp EcoRI fragment of the rat a subunit of the high-affinity IgE receptor (clone Edp 16)*3; myeloperoxidase,

From The Walter and Eliza Hall Institute of Medical Research, Post

Submitted July 23,1991; accepted October 16, 1991. Supported by the National Health and Medical Research Council of

Australia and the US National Cancer Institute (CA12421). A.G.E. is a National Health and Medical Research Council Postgraduate Scholar.

Address reprint requests to Suzanne Cory, PhD, The Walter and Eliza Hall Institute of Medical Research, Post Ofice Royal Melbourne Hospital, Victoria, 3050, Australia.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section I734 solely to indicate this fact.

office Royal Melbourne Hospital, victoria, Australia.

0 I992 by The American Society of Hematology. OOO6-4971/92/ 7905-0Oo9$3.OO/O

Blood, Vol 79, No 5 (March 1). 1992: pp 1271-1281 1271

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1272 ELEFANTY AND CORY

the 1.9-kb Pst I fragment of the murine cDNA clone mMP06”; p53,0.4-kb and 0.87-kb Pst I fragments of the murine cDNA clone pp53-176=; and scl, the 1.9-kb EcoRI fragment of the murine cDNA, corresponding to nucleotides 406 to 2335.% The origin of other probes is described el~ewhere.’~~~’ Probes were labeled with [a-”PIdATP using a random hexamer priming kit (Bresatec Lim- ited, Adelaide, South Australia).

Hematopoietic Assays Conditioned medium collected from dense cell cultures was

concentrated 10-fold in an ultrafiltration cell (model 8050; Amicon, Danvers, MA) and filtered before use. Granulocye-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were assayed using FDC-PI cells (responsive to GM-CSF and IL-3) and 32D clone 23 cells (responsive to IL-3) as described previously.” Erythropoietin (Epo) was assayed on fetal liver cells using day-2 colony-forming unit-erythroid (CFU-E) as a read0ut.2~ Levels of CSF were converted to bone marrow units’” by comparison with purified recombinant GM-CSF and IL-3 of known activity. Colony- forming cells were assayed by culturing cell lines at a range of concentrations in triplicate 1 mL agar cultures, with and without addition of indicated growth factors for 7 to 14 days at 37°C as described previously.” Recombinant murine IL-3, GM-CSF, granulocyte-CSF (G-CSF), and leukemia inhibitory factor (LIF) were generously provided by Dr D. Metcalf, and recombinant human Epo by Dr A.W. Burgess.

Detection of Cell Su~ace Antigens Cells were stained with the monoclonal antibodies (MoAbs)

M1/70 (aMac-l),SZ RB6-8C5 (‘8C5’/aGr-l),” F4/80,” and TER- 119”; each had been directly conjugated to fluorescein isothiocy- anate (FITC) or phycoerythrin (PE) (Southern Biotechnology Associates, Birmingham, AL), or biotinylated and revealed by an FITC- or PE-streptavidin second stage. All staining was performed in the presence of excess unlabeled anti-Fcy receptor antibody 2.4G2% to reduce nonspecific binding of antibodies, and cells were analyzed on a FACScan (Becton Dickinson, Sunnyvale, CA).

Cytochemical Stains

May-Grunwald-Giemsa, Lux01 Fast Blue, Astra Blue, and staining for a-naphthyl acetate esterase, myeloperoxidase, and acetylcholinesterase were performed on cytocentrifuge preparations or fixed agar colonies according to standard protocol^.^' Detection of the pseudoperoxidase activity of hemoglobin in agar colonies or cell suspensions was with benzidine HCl” or 2,7-diaminofl~orene.’~

RESULTS

Erythroid and mixed erythroid/mast cell hyperplasia was a frequent cause of morbidity in mice we reconstituted with bcr-abl virus-infected bone marrow cellsI9 (Elefanty and Cory, manuscript submitted). To further characterize this syndrome, we sought to establish cell lines by culturing the bone marrow and spleen cells of 19 mice in the absence of added growth factors. Twelve of the 15 lines that were derived (Table 1) were mast cell in origin; three grew rapidly from the outset but the majority grew very slowly, doubling every 2 to 7 days. The proviral insertion pattern shown by Southern blot analysis established that all but one of the cell lines were clonal and, in 9 of 10 cases, the pattern obtained from the cell line was identical to that obtained from the tissue of origin. Because six of the seven mast cell

lines in this analysis derived from tissue infiltrated predom- inantly by erythroid cells, both populations probably descended from a common infected prec~rsor.’~

Six mast cell lines were maintained continuously in vitro for several months. Remarkably, they proved to be pheno- typically unstable (Table 2), most evolving towards erythroid/megakaryocytic differentiation. After cloning in agar, the phenotypic variants from all six lines were shown by proviral insertion analysis to derive from the same infected precursor as the initial mast cell line” (this report and unpublished results). The history of one erythroid/ megakaryocytic line (96.4) will be described in detail, as will that of an exceptional line (34.6) that became myeloid. The evolution of the bcr-ab1 lines towards a fully malignant phenotype will also be considered.

The 96.4 Line: A Switch From Mast Cell to Erythroid and Megakaryocytic Differentiation

Clonal phenotypic variants. Figure 1A summarizes the evolution of the 96.4 mast cell line, which was obtained from the spleen of a BALB/c mouse with erythroid, mast cell, and macrophage disease (Elefanty and Cory, manuscript submitted). The slow-growing line was independent of the exogenous IL-3 required by normal mast cells, but may have been provided with growth factors by the stromal component present during the first few weeks of culture. After about 3 months, a rapidly growing subline of mast cells (designated 96.43Mst) arose in one of several cultures grown in parallel. The cells in another flask of the slow- growing line (designated 96.42Mst) underwent a dramatic change after another 2 months. Within 1 week, the culture became pleomorphic (compare Fig 1B with 1C and D) and cytospin preparations (Fig 1E and F) showed that the mast cells had been replaced by maturing erythroid cells and megakaryocytes that stained with acetylcholinesterase (not shown). Most cells (70%) in this subline (96.42EMeg) contained hemoglobin, as judged by staining with benzidine or diaminofluorene and over 50% reacted strongly with the erythroid-specific MoAb TER-119.35 After 2 months, one 96.42EMeg culture yielded a subline (96.42Ery) that retained erythroid character but exhibited few mature megakaryocytes.

Evidence that the mast cell, erythroid/megakaryocytic, and erythroid subclones all carried the same proviral insert as the original splenic tissue and hence derived from a single infected precursor was provided by Southern blot analysis for provirus-host DNA junction fragments in EcoRI digests (Fig 2A) and Hind111 digests (not shown). RNA analysis (Fig 2B) established that the bcr-ab1 gene was expressed and confirmed the lineage assignments of the cell lines. Expression of the a chain of the high-affinity IgE receptor’’ and c-kit, the receptor for mast cell growth factor (MGF)? was detectable only in the mast cell lines (lanes 1 and 2), while p globin and Epo receptor expression was found only in sublines containing erythroid cells (lanes 3 and 4). Two genes encoding transcription factors impli- cated in erythroid differentiation, GATA-1 and scl/tal, were expressed in the mast, erythroid, and erythroid/




bcr-ab/ MAST CELLS SWITCH DIFFERENTIATION 1273

Table 1. Derivation of Cell Lines From Primary bcr-ab/ Tumors

Primary Disease

Latency Clonal Mouse Strain (wk)* Pathologyt Transplantation* Cell Llne5 Relationship11

33.3 C57BL/6 46 Ery, Mst + ** Erythroid [f] I 55.3 BALB/c 22 Ery, Mst ND Erythroid [fl I 52.10 BALB/c 30 Ery, Mst +tt Erythroid [f] I 34.4 C57BL/6 49 Mst + Mast [fl I 45.14 DBA/2 16 Ery, Mst + Mast [fl I 99.4 BALB/c 24 Ery + ** Mast [fl D 32.1 C57BL/6 8 Ery, Mst - Mast [SI ND 33.1 C57BL/6 7 Ery Mast [SI ND 33.2 C57BL/6 7 Ery, Tn Mast [SI ND

- Mast [SI ND 34.6 C57BLl6 33 Ery 34.8 C57BLl6 22 Tll Mast [SI I 45.1 1 DBA/2 3 ErY -tt Mast [SI I 96.4 BALBIc 11 Ery, Mst +# Mast [SI I 52.5 BALB/c 11 Ery, Mst Mast [SI I 59.17 BALB/c 14 Ery, Mst ND Mast [SI I

-

-

-

-

Abbreviation: ND, not done. *Time elapsed (in weeks) between transplantation of irradiated mouse with bcr-ab/ virus-infected bone marrow cells and autopsy. tHistopathology of primarybcr-ab/ mice that exhibited erythroid (Ery) and mast cell (Mst) diseaselg (Elefanty and Cory, manuscript submitted). Two

of these mice also had a lymphoid (T) tumor component and all had a variable amount of macrophage tumor that, at least in some cases, derived from the same infected progenitor as the erythroid/mast cell t ~ m o r . ’ ~

STransplantability of the primary tumor was usually assessed by injecting lo6 spleen cells intraperitoneally into 2 to 6 sublethally irradiated (4.5 Gy) or nonirradiated (two daggers) recipients, except for those (two asterisks) that were tested as cell lines within 25 days of initiating in vitro culture. Recipients were monitored for tumors for 12 months; +,transplantable; -, not transplantable.

§Cell type in primary line derived by culturing spleen or bone marrow cells of sick mice. [fl or [SI indicates fast or slow growth. IlRetroviral integration pattern of cell line determined by Southern blotting (see Fig 2A) to be identical (I) or different (D) to that of tissue of origin or,

for mouse 34.8, of tumor deposits elsewhere in the primary mouse. ND, not done due to insufficient material; these four animals had oligoclonal tumors at other sites.

llA clonal T-lymphoid thymoma was present that derived from the same progenitor (mouse 33.2) or a different progenitor (mouse 34.8) to the macrophage or mast cell disea~e.’~

#Four of six recipients developed tumors following a long latency (6 months).

Table 2. Phenotypic Shifts of bcr-ab/ Mast Cell Lines

Cell Line*

96.4Mst 34.6Mst

34.4Mst 45.14Mst 52.5Mst 99.4Mst

Culture Period (mo)t

5 3

12 6

1 II 1.511

6 6

Secondary Lineage*

Erythroid/megakaryocytic Erythroid/mast cell Erythroid§ Myeloid§ Erythroid/megakaryocytic Erythroid/megakaryocyticT Erythroid/megakaryocytic# Erythroid/mast cell

*Six other mast cell lines did not demonstrate a phenotypic shift in

tTotal period in culture (in months) before the observed shift. Sln all cases, agar cloned cell lines of the secondary lineage harbored

identical proviral integrations to the primary cell line (Figs 2 and 5, Elefanty et aI.,”and data not shown).

vitro, but they were only cultured for 1 to 4 months.

§These lines evolved from the erythroid/mast cell line. IlAlthough mast cells dominated the primary cultures from these

mice, some erythroblasts persisted from the outset. BPreviously described,” but the megakaryocytic component was not

initially appreciated. #Rare clones obtained after infection of the 52.5Mst line with a

retrovirus expressing a mutated p53 cDNA; whether p53 played any role is obscure.

megakaryocytic variants, consistent with previous observa- t i o n ~ . ~ ’ . ~ ~

Four of five 96.4 mast cell sublines expressed GM-CSF RNA (eg, Fig 2B, lane 2) and, although no IL-3 (or MGF) RNA was detectable, more sensitive bioassays (see Materials and Methods) showed that the three clones tested secreted low levels of IL-3 (1 to 5 U/mL) as well as GM-CSF (5 to 900 U/mL). All three erythroid/megakaryocytic and both erythroid subclones tested secreted GM-CSF (5 to 100 U/mL). A low level of Epo bioactivity (0.05 U/mL) was detected in the single erythroid clone tested, but not in an erythroid/megakaryocytic clone.

The range of differentiation stages detectable in the 96.42EMeg and 96.42Ery lines was impressive. Both produced macroscopic colonies of erythroblasts in the spleen of lethally irradiated syngeneic mice 12 to 14 days after injection. Spleen colony formation, a property displayed by some early stem cells (CFU-S), was not obtained with the other bcr-ab1 erythroid cell line (33.3Ery) tested. When cultured in semi-solid agar, the 96.42 erythroid lines yielded diverse colony types (Table 3). The EMeg line produced large compact colonies containing blast cells (some colonies were white, others red due to a large erythroid component), as well as small single-

Factor production.

Differentiation capacity of the 96.4 variants.





MOUSE 96.4 96.42EMeg c erythroid, mast &

tumor \

- macrophage spleen (SIOW)

culture

Fig 1. Differentiation of 96.4 cell lines. (A) In vitro history (see text). The interval between stages (boxed) is indicated in months. (B through D) Appear- ance in culture of (B) 96.42Mst (8 months) (C) 96.42EMeg

* (5 months) (original magnification of both x 125). and (0 ) mega- karyocyte in 96.42EMeg culture (original magnification x400). (E through G) Cytocentrifuge preparations stained with May-Grun- wald-Giemsa of (E) 96.4Mst showing granulated mast cells (5 weeks) and IF) 96.42EMeg showina meaakarvocvte and iY

lineage or mixed colonies of maturing erythroid cells and megakaryocytes (Fig 3). In contrast, the Ery subline yielded fewcr megakaryocytic colonies and many more overtly hemoglobinized colonies (Table 3). Only the white compact colonies retained a high proliferative potential upon replat- ing (data not shown). Addition to the agar cultures of IL-3 or Epo (Table 3) or GM-CSF (not shown) affected neither the plating efficiency nor the distribution, size, or morphology of colonies.

The 96.4 line did not readily switch its differentiation pattem. Despite the phenotypic shift just described, the 96.4 primary mast cell line was not intrinsically unstable in vitro. After being maintained for 8 months in liquid culture, 96.42Mst cells could be cloned in agar, but only on onc occasion over the next 5 months were erythroid colonies detected amongst the mast cell colonies. These were established as EMeg lines that displayed the same proviral insert as all other 96.4 sublines (Fig 2A, lane 5), but the liquid culture from which they derived was not overtaken by erythroid cells, perhaps because the mast cells themselves now grew more rapidly or because the second EMegvariant was not as competitive as the earlier one.

Because the demethylating agent 5-azacytidine has been

" "- , , . .

erythroid differentiation with maturing normoblasts and an erythrocyte (arrowed) (5 months) (original magnification x500). (G) Peripheral blood film from an erythroleukemic recipient of 96.42EMeg (5 months) (original magnification x500). Note the maturity of the circulating erythroblasts. The time indicated in parentheses in this and all subsequent figures refers to the total period in culture at the time of analysis.

shown to induce a lymphoid to macrophage switch in an Abelson-transformed ccll line,M we attempted to induce a mast to erythroid/megakaryoqtic switch by incubating three 96.42 Mst clones with up to 6 pmol/L 5-azacytidine for 1 to 5 days. As only one treated culture acquired a small number ( - 12%) of benzidine-staining cells, we infer that the genetic change(s) required to permit erythroid/ megakaryocytic differentiation of the 96.42Mst progenitor cell is not readily induced by demethylation.

The 34.6 Line: A Switch From Mast Cell to Erythroid and Myeloid Differentiation

The 34.6 mast cell line (34.6Mst) was derived from the near-normal bone marrow of a C57BL/6 mouse with a modest hcr-ahl-induced erythroid and macrophage tumor burden." As outlined in Fig 4A, prolonged culture of this line produced both an erythroid and a myeloid variant. Once again, Southern blotting established that the subcloned phenotypic variants and the primary mast cell line derived from a single infected precursor (Fig 5A).

After 3 to 4 months in culture, the cells became smaller, generally exhibiting a morphology intermediate between that of an erythroid and

Evolution of the erythroid variant.




bcr-ab/ MAST CELLS SWITCH DIFFERENTIATION

B

1275

Table 3. Colony Formation by 96.42 Erythroid Lines in Agar

A 1 2 3 4 5 6

/ / I \ \ \ SpI Mast EMeg Ery

C

bcr-abl

IgE-R

13 globln

Epo-R

GATA-1

GAPDH

SCl

LTR R R LTR C-klt

GYCSF

GAPDH

1 2 3 4

S f / T I \ Mast EMeg Ery

Fig 2. Molecular analysis of 96.4 cell lines. (A) Southem blot analysis with probe 8 (see C) of EcoRI-digested DNA (15 pg) from (1) spleen of mouse 96.4, (2) 96.4Mst (3 weeks), (3) 96.42Mst.3 (9 months), (4) 96.42EMeg.2 from initial switch (5 months), (5) 96.42EMeg.4 from agar culture of 96.42Mst (9 months) (see text), and (6) 96.42Ery.3 (11 months). The examples shown were nonadjacent tracks from the same blot. (B) Northern blot analysis of polyadenylated RNA (2 to 4 pg) from (1) 96.4Mst (1 month), (2) 96.43Mst (6 months), (3) 96.42EMeg (6 months), and (4) 96.42Ery (8 months). The examples shown were nonadjacent lanes from two separate blots sequentially hybridized with the indicated probes (see Materials and Methods), GAPDH hybridization providing a control for RNA loading. The nomenclature for the lines used in this and subsequent figures includes the number of the primary mouse, an abbreviation of the phenotype, and a period followed by a clone number if the line was subcloned in agar (eg, 96.42EMeg.2). Abbreviations used here and in subsequent figures: Spl, spleen; Mast, mast cell line; EMeg, erythroid/ megakaryocytic cell line; Ery, erythroid cell line; 's,'slow growing; 'f,' fast growing; IgE-R, high affinity IgE receptor; Epo-R, Epo receptor. The bcr-ab/ transcript was detected by a 1.4 kb bcr probe.19 (C) Structure of the MPZen(bCr-8bn provirus." The vector sequences derive from Moloney leukemia virus except for the hatched region, which is from the myeloproliferative sarcoma virus. The location of probe a, a 360-bp Xho I-Hincll fragment that derives from the p15E region of Moloney virus is indicated, as are restriction sites for EcoRl (R) andHindlll (HI, and the retroviral long terminal repeats (LTR).

a mast cell (34.6EMst) (compare Fig 4B with C), although occasional cells resembled myelocytes and monocytes. RNA analysis showed messenger RNAs (mRNAs) for both p globin and the mast cell-specific IgE receptor a chain, as well as c-kit (Fig 5B, lane 1). At this point the cell line was cloned in agar and transplanted (see below). With time, 34.6EMst subclones assumed further erythroid character (compare Fig 4D with C), with increased expression of p globin and a loss of expression of the IgE receptor and c-kit (compare lanes 1 and 2 in Fig 5B). Thc clone subsequently analyzed in detail (34.6Ery.3) formed agar colonies contain-

Colony Morphology (%):

White Red Megakaryocytic, Cell Line' Stimulust Compact Compact Mixed5 Erythroid Diffuse11

96.42EMeg Saline 2 9 70 18 1 IL-3 4 9 64 21 2 EPO 2 13 71 13 1

96.42Ery Saline 7 34 13 4 6 - IL-3 5 31 23 41 -

27 50 - 6 17 EPO

"The 96.42EMeg cell line was assayed after 6 months and the

tlL-3 was used at 1,000 UlmL and Epo at 1 UlmL. *Morphology after 7 days of culture; 150 to 500 colonies from 4 to 9

plates were scored per stimulus. The plating efficiency of 96.42EMeg ranged from 0.7% to 0.9% and of 96.42Ery from 0.4% to 1.3% for the different stimuli, and was linear over cell concentrations of lo' to 1O'lmL. Agar plates and individual colonies were stained with benzidine or for acetylcholinesterase to confirm erythroid and megakaryocytic elements, respectively. White compact colonies were composed of blast cells with small numbers of erythroblasts and megakaryocytes, while red compact colonies were dominated by erythroblasts with few blasts and megakaryocytes.

§Colonies containing megakaryocytes, either alone or mixed with erythroid cells.

I!Colonies with a diffuse morphology (Fig 3F) that contained erythroid and megakaryocytic cells.

96.42Ery line after 8 months in culture.

ing benzidine-positive cells in the absence of added growth factors, but the degree of erythroid maturation was mark- edly less than in the 96.42EMeg line and megakaryocytic differentiation was absent. Ncither plating efficiency nor colony morphology was affected by inclusion of IL-3, Epo, LIF, GM-CSF, G-CSF, or M-CSF, but 1.5% dimethyl sulfoxide (DMSO) completely inhibited colony formation.

Immunophcnotyping (not shown) showed that approxi- mately 5% of the cells in 34.6Ery.3 clones expressed high

Fig 3. Colony morphology in cultures of 96.42EMeg grown in agar for 7 days. (A) White compact, (B) red compact, (C) erythroid, (Dl megakaryocytic, (E) mixed erythroid/megakaryocytic, and (F) diffuse colonies (original magnification x50) (see text and Table 3).




ELEFANTY AND CORY 1276

cloned 34.6Ery + A

MOUSE 34.6 erythroid & macrophage BM 3 mo

tumor

34.6MSt 34.6EMst

34.6Myl + culture

Fig 4. Differentiation of 34.6 cell lines. (A) In vitro history (see text). (B through D) Cytocentri- fuga preparations stained with May-Griinwald-Giemsa (MGG) of (B) 34.6Mst (2 months), (C) 34.6EMst (3.5 months), and (D) 34.6Ery (12 months) (original magnification x 500). Appearance in culture of (E) 34.6Ery (12 months) and (F) 34.6Myl (14 months) (original magnification x 125). Cytocentrifuge preparations of 34.6Myl stained (G) with MGG, (H) for myeloperoxidase, ( I ) with MGG after 2 days of induction with 1.5% DMSO, and (J) with MGG after 24 hours of treatment with 16 nmol/L tetradecanoyl phorbol acetate and overnight incubation with latex beads (0.8 pm; Difco, Detroit, MI) (original magnifications: G, H, 1, x500; J, x800).

levels of TER-119. Curiously, most cells expressed very high levels of F4/80, an antigen not seen in the 96.42EMeg line and previously thought to be macrophage although other features suggestive of macrophage diffcrcnti- ation were not present. Attempts to differentiate 34.6Ery.3 cells with 1.5% DMSO in liquid culture showed marked toxicity, but the surviving cells acquired some cytoplasmic granules suggestive of reversion to a mast cell phenotype.

After 2 months in vitro, the mast cell culture generated from the spleen of a mouse transplanted with 34.6EMst cells was monopolized by maturing myeloid cells that grew in tight clumps (Fig 4F), unlike the crythroid/mast ccll or its erythroid derivative (Fig 4E). The line was cloned in agar and designated

A shft to myeloid differentiation.

cytic cells (Fig 4G) that stained strongly for myeloperoxidase (Fig 4H). Depending on the clone, 5% to 80% of the cells were cosinophils and their precursors, because Lux01 Fast Blue showed small numbers of emerald green cytoplasmic granules. Less than 5% of the cells were phagocytic adherent macrophages but - 30% expressed a-naphthyl acetate esterase. RNA analysis showed myeloperoxidase transcripts in 34.6Myl but no detectable mRNA for p globin or the IgE receptor (Fig 5B, lane 3).

34.6MyI cells formed granulocyte-macrophage colonies in agar in the absence of added growth factors. Neither plating efficiency nor colony morphology was affected by the inclusion of IL-3, GM-CSF, G-CSF, M-CSF, Epo, or LIF in the assays (not shown). However, analysis of

34.6Myl. All 12 clones analyzed were dominated by granulo- conditioned medium showed endogenous production of





A 1 2 3

6.5kb ww-w

/ I \ Mast E h My1

1 2 3 4 5 6

bcr-abl 11) @a (c

13 globin

C-kit

MPO

GM-CSF

GAPDH

IgE-R

GAPDH

111 - -

a

Ilo - * // - + + +

EMast Ery Myl +DMSO

Fig 5. Molecular analysis of 34.8 cell liner. (A) Southem blot analysis with probea (see Fig 2C) of EcoRI-digested DNA (15 pg)from (1) 34.6Mst (2.5 months), (2) 34.6Ery.3 (16 months), and (3) 34.6Myl.l (16 months). (B) Northem blot analysis of polyadenylated RNA (2 to 4 pg) from (1) 34.6EMst (3 months), (2) 34.6Ery.3 (13 months), (3) 34.6Myl.l (15 months), and (4 through 6) 34.6Myl.l incubated with 1.5% DMSO for 1,3, or 5 days (16 months). The examples shown were nonadjacent lanes from two separate blots sequentially hybridized with the indicated probes (see Materials and Methods), GAPDH hybridization providing a control for RNA loading. Abbreviations as in Fig 2 and Myl, myeloid cell line; EMst, erythroid/mast cell line; MPO, myeloperoxidase.

GM-CSF (up to 1,400 U/mL) and IL-3 (up to 100 U/mL). In contrast, minimal levels of GM-CSF were detected in the 34.6 mast and erythroid phenotypic variants.

Granulocytic differentiation of 34.6Myl cells was enhanced by DMSO and sodium butyrate (Fig 41), and 16 nmol/L tetradecanoyl phorbol acetate increased the number of adherent phagocytic macrophages in some clones (Fig 45). DMSO increased Mac-1 expression and most cells (>90%) acquired F4/80, while a subpopulation (5% to 15%) also expressed the 8C5 mature granulocytic marker (data not shown). Granulocytic differentiation was associated with downregulation of myeloperoxidase, as assessed by RNA analysis (Fig SB, lanes 3 through 6) and cytochemical staining (not shown), and also with considerable GM-CSF mRNA induction (Fig 5B).

Evolution of the Transformed Phenotype The slowly growing mast cell lines were not malignant

(Table 4) and, with one exception, were derived from animals in which the primary disease, although lethal, was not transplantable (Table 1). Furthermore, although at 18

weeks the still slowly growing 96.42Mst culture contained transplantable cells, the tumor latency was very long (37 weeks) and only after 29 weeks in culture were clonogenic cells detectable in agar. These observations suggest that although bcr-ab1 expression confers a growth advantage in vivo and in vitro, further somatic mutation(s) must accrue to achieve complete transformation. Those cell lines (erythroid or mast) that were transplantable from the outset (Table 4), and that derived from transplantable primary tumors (Table l), had presumably already acquired secondary mutation(s) in vivo.

Because the tumor suppressor gene p53 is mutated in many CML patients in blast crisis:d7 we investigated p53 expression in bcr-ab1 lines that had become malignant. Diminished expression of p53 may have contributed to the malignancy of the 33.3Ery line, because p53 RNA levels were much lower in this line, especially after prolonged culture (Fig 6C, lanes 13 and 14), than in other erythroid lines (Fig 6, lanes 3, 4, 6, 12, 15, and 16). Levels were also low in the myeloid derivative of 34.6 (Fig 6B) and in three of the four mast cell lines examined (Fig 6, lanes 1,2, and 10). Sequence analysis will be required to fully assess the role of p53, because oncogenic changes affecting p53 function frequently involve point mutations" and thus may not be detectable by Northern analysis.

The ets-related transcription factor PU.1, expressed in macrophage and B-lymphoid cells,@' corresponds to a gene previously designated Spi-1 or Sfpi-1 that is silent in normal erythroid cells but frequently activated by proviral insertion in Friend erythroleukemia.m Sfpi-1 expression was readily detectable in several of the erythroid lines (Fig 6, lanes 6, 12,13, and 15) and may therefore have played a role in their neoplastic progression. Other bcr-ab1 erythroid lines, however, lacked Sfii-1 expression (Fig 6, lanes 3,4, and 16).

Differentiation Capacity of the Cell Lines In vivo Histologic analysis of animals transplanted with the

established cell lines permitted assessment of their differentiation capacity in vivo (Table 4). Mice injected with 96.42EMeg cells developed erythroleukemia with greatly elevated numbers of circulating erythroblasts (Fig 1G). Although both 96.42Mst and 96.43Mst cells produced disseminated mast cell tumors, all four animals examined also contained areas of erythroid tumor in the spleen. This finding may indicate that the cells had a wider differentiation potential in vivo than in vitro, or that the frequency of 'lineage switch' mutations was relatively high in vivo. Within the 34.6 series, the 34.6Myl.l clonc produced disseminated myelomonocytic tumors, while all six recipients of 34.6EMst cells succumbed to various combinations of erythroid, mast cell, and macrophage tumors. Unexpect- edly, the tumors arising after injcction of the relatively undifferentiated erythroid clone 34.6Ery.3 were mast cells, implying that its differentiation potential had also been modified by environmental signals or by mutation.

DISCUSSION

The bcr-abl-expressing mast cell lines characterized here were obtained from mice that had developed erythroid and/or mast cell hyperplasia after lethal irradiation and





Table 4. Clonogenicity and Transplantability of bcr-abl Cell Lines

Transplanted Tumors:

Culture Period Clonogenicity Latency Cell Line (wk)' (%)t Incidence5 lwk)ll Pathologfl

96.4 lines - - 96.4Mst Is] 5 ND 014

96.42Mst Is] 18 213 37 Mst(Ery) 23.29 < 0.002

33 15 96.43Mst [fl 14 ND 313 8 Mst(Ery) 96.42EMeg 24 1 213 11 Ery#

313 (IV) 4 EW 34.6 lines

34.6EMst 15 616 8 Mst, Ery, Mac** 17 10

34.6Ery.3 51 313 7 Mst 56 15

34.6Myl.l 50 45 66 1 /3 44 My1

2/3 (IV) 8 My1 Other lines

- - 33.1Mst Is] 8 ND 013 52.5Mst Is] 5 0/3 - -

8.26 <0.01 42 20

7 5 99.4Mst [q 4 313 3 Mst

33.3Ery 2 1 313 3 EryWst)

Abbreviation: ND, not done. 'Time of continuous culture (in weeks) before transplantation or plating in agar; Is] and (q indicate slow and fast growing mast cell lines. t u p to 10'cells (5 x 10'for 96.42Mst cells) plated in triplicate 1 mL agar cultures were scored for colonyformation at day 7 and 14, and the cloning

efficiency expressed as a percentage of the cells plated. No colonies were observed for 96.42Mst before 33 weeks of culture or for 52.5Mst before 42 weeks of culture. Equivalent results were obtained for mast cell lines cultured with or without IL-3 (1,000 UlmL).

SCeils (lo8) were transplanted intraperitoneally into sublethally (4.5 Gy) irradiated recipients, or nonirradiated mice (boldface), and monitored for up to 12 months. Additional animals were inoculated intravenously (IV) where indicated.

§Number of mice developing tumors versus total number injected. IlMean tumor latency (in weeks) posttransplantation. THistopathology of secondary tumors: Mst, mast cell; Ery, erythroblast; Mac, macrophage; Myl, myeloid. Minor tumor comDonents are indicated

#One of two recipients also had mast cell tumor around the common bile duct. **Previously described."

in parentheses (see text).

A 96.4 lines

1 2 3 4

P53

SfpC1

GAPDH m m w

S 't/ I \ Mast EMeg Ery

B 34.6 lines

5 6 7 8 9

I I + + + EMast Ery Myl+~hlSO

C

10 11 12 13 14 15 16

-e/-- ----- / / I \ I \ \

Mast EMeg Ery EMeg Ery

Fig 6. Northern blot analysis of cell lines harboringbcr-ab/ provirus. Polyadenylated RNA samples (2 t o 4 pg) were sequentially hybridized with the indicated probes (see Materials and Meth- ods). Samples for (A) are as for Fig 28. In (E), (5) 34.6EMst (3 months), (6) 34.6Ery.3 (13 months), and (7 through 9) 34.6Myl.l incubated with 1.5% DMSO for 1, 3, or 5 days (16 months). In (C), (10) 52.5Mst (3 months), (11) 99.4Mst (5 weeks), (12) 45.14EMeg.2 (14 months), (13) 33.3Ery (1 week), (14) 33.3Ery.2 (11 months), (15) 34.4EMeg (7 months), and (16) 55.3Ery (2 weeks).





reconstitution with hcr-ah1 virus-infected bone marrow" (Elcfanty and Cory, manuscript submitted). The virus- infected cell responsible for the syndrome appears to have been a progenitor cell with capacity for both mast cell and erythroid differentiation, as judged by clonal analysis of affected tissues and derived cell lines (Elcfanty et all" and this report). The hcr-ah1 mast cell lines grew in the absence of exogenous IL-3, unlike normal mast cells, but initially may have required factors produced by associated stromal cells, eg, the recently identified MGF or Steel factor. They produced significant levels of GM-CSF and IL-3. When first established, most lines were neither transplantable nor clonogenic in soft agar, but transplantable cells emerged after prolonged culture, prcsumably due to additional (undefined) oncogenic mutations. Analogous factor-indc- pendent mast cell lines produced by Abelson virus infection of multilineage or blast cell colonies"." or 18-day fetal liver cells" also secreted multiple growth factors"." and initially required stromal cell support,"." but became fully malignant much more rapidly than the hcr-ah1 lines (2 to 3 months after virus infcction v 6 to 20 months). This finding probably reflects differences in the transforming potency of v-uhl and p210 hcr-ah1 proteins.

The striking feature of the hcr-ah1 cell lines was their propensity to spontaneously change hematopoietic lineage on prolonged culture, a property never previously docu- mented for mast cell lines. Several assumed an erythroid or erythroid/megakaryocytic character, but one became myeloid (Table 2). In each case, the variant line contained the same proviral insert as the parental mast cell line (Fig 2A and SA and data not shown).

Several of the variant sublines exhibited a marked capacity to continuously differentiate, despite being highly malignant. The 96.4EMeg line and its subclones, for example, yielded mature erythrocytes and megakaryocytes in the absence of added growth factors (Fig 1). Because normal erythroid differentiation requires Epo and/or IL-3, and Epo was detected in the conditioned medium of one hcr-ah1 erythroid clone, an autocrine loop may have figured in their evolution. Intriguingly, spontaneous hemoglobinization is a feature of some erythroid progenitors in the chronic phase of CML.'6 A surprisingly high proportion (5 of 13) of the cell lines established from CML blast crisis patients (eg, K562) exhibit some erythroid and/or megakaryocytic character, but induction by chemical agents is usually required to promote differentiation."' Equivalent lines have not been isolated with Abelson virus; differentiating erythroid colonies isolated from infected fctal liver cells could not be established in culture."

The differentiation capacity of the hcr-ah1 myeloid line (34.6Myl) strongly resembled that of the well-studied promyelocytic line HL-60 derived from a patient with acute myeloid leukemia.'" Both differentiate spontaneously along neutrophil, eosinophil, and macrophage lines, the degree of differentiation being enhanced by a range of chemical inducers. 34.6Myl cells secrete GM-CSF and IL-3 and an 'autocrinc activity' has been observed in H L d k o n d i - tioned medium.5N None of the existing CML lines shows such a degree of myeloid differentiation and no equivalent

lines have been isolated with Abelson virus. However, one clone of the IL-3-dcpcndcnt line 32D, which was initially characterized as a mast cell line,'" has the capacity to terminally differentiate into granulocytes if IL-3 is replaced by G-CSF."'

The nature of the cells involved in the 'lineage switches' is an intriguing, although unresolved, issue. Figure 7 depicts our current working hypothesis It seems unlikely that mature mast cells are capable of the phenotypic shifts observed, so we presume that the affected cells were precursors present at low concentration in the cultures. These cells may have been committed, unipotential mast cell progenitors or oligopotential cells unable to express their full potential under the growth conditions provided. (The observation that certain lines exhibited a wider differentiation potential in vivo than in vitro may favor the latter hypothesis.) In either case, phenotypic switching in vitro prcsumably resulted from somatic mutation(s) that either provided necessary growth factor(s) or induced key transcriptional programming gene(s). Our failure to mimic such mutation by treatment with 5-azacytidinc suggests that the mechanism of the lineage switch is more complex than activation of a single gene by demethylation.

For 96.42, the switch from mast to erythroid/megakaryocytic differentiation was accompanied by transformation, raising the possibility that the same gene(s) orchestrated both events. Many of the genes encoding nuclear oncopro- tcins (which might cooperate with cytoplasmic hcr-ahl) are known to be transcription factors and might well influence the differentiation program. However, transformation and phenotypic change were not always linked. The myeloid switch in the 34.6 line, for example, occurred in a cell that was already tumorigenic and clonable in agar.

The evolution of the hcr-ahl-cxpressing cell lines pro-

Progenitor cells

mutation/ \ mutation

Mast cell differentiation

Erythroid/megakaryocytic Myeloid differentiation differentiation

Fig 7. Differentiation pathways in bcr-ab/ mast cell lines and their phenotypic variants. This model suggests that mutationfs) in mast cell progenitors 'switch' differentiation either to erythroid and megakaryocytic or to myeloid lineages. The mast cell precursors are cells with self-renewal potential clonally descended from the bcr-ab/ virus-infected oligopotential progenitor that caused disease in the primary mice. These cells may be unipotential or they may retain oligopotentiality but be unable to express it in vitro.





vides clues to the function of bcr-ab1 and its role in CML. Expression of bcr-ab1 by a hematopoietic progenitor and its progeny provides only a modest proliferative advantage and relieves certain growth factor requirements but probably not ail. Whether growth factor production, particularly of IL-3,I3 is crucial to the action of bcr-ab1 (and v-abl) or is merely an epiphenomenon remains an important unresolved issue. The unexpected phenotypic plasticity of the cell lines suggests that bcr-ab1 expression may facilitate and/or complement the (postulated) somatic mutations in lineage-determining genes. Finally, our observation that even fully transformed lines can retain substantial capacity for maturation suggests that the chronic phase of CML may accumulate mutations that increase malignancy without significantly curtailing differentiation. Thus, more events

may be needed to progress to blast crisis than hitherto suspected. The lines characterized here may facilitate identification not only of genes able to cooperate with bcr-ab1 in leukemogenesis but also those instrumental in determining lineage commitment during normal hematopoi- esis.

ACKNOWLEDGMENT

We thank Drs J.M. Adams, A.W. Harris, D. Metcalf, and N.M. Gough for helpful discussions and critically reviewing the manuscript, Dr T. Kina for providing the TER-119 MoAb, Dr J. Visvader for the scl probe, Dr G. Rovera for the myeloperoxidase probe, Dr J.P. Kinet for the IgE receptor probe, Dr G.R. Johnson for performing the Epo bioassay, and Dr A. Strasser for providing reagents and helpful advice for flow cytometry.

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8)




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